Methods of stratifying patients for treatment with retinoic acid receptor-alpha agonists

ABSTRACT

The invention provides methods that define cellular populations that are sensitive to RARA agonists and identify patient subgroups that will benefit from treatment with RARA agonists. The invention also provides packaged pharmaceutical compositions that comprise a RARA agonist and instructions for determining if such agonist is suitable for use in treatment.

CLAIM OF PRIORITY

This application is a division of U.S. patent application Ser. No.15/899,629, filed Feb. 20, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/799,519, filed Oct. 31, 2017, now U.S. Pat. No.10,167,518, which is a continuation of U.S. patent application Ser. No.15/172,045, filed Jun. 2, 2016, now U.S. Pat. No. 9,845,508, which is acontinuation of International Application No. PCT/US2016/025256, filedMar. 31, 2016, which claims the benefit of U.S. Provisional ApplicationNo. 62/268,203, filed Dec. 16, 2015, and U.S. Provisional ApplicationNo. 62/140,999, filed Mar. 31, 2015. The contents of each of theforegoing applications are incorporated herein by reference in theirentirety.

BACKGROUND OF THE INVENTION

Retinoids are a class of compounds structurally related to vitamin A,comprising natural and synthetic compounds. Several series of retinoidshave been found clinically useful in the treatment of dermatological andoncological diseases. Retinoic acid and its other naturally occurringretinoid analogs (9-cis retinoic acid, all-trans 3,4-didehydro retinoicacid, 4-oxo retinoic acid and retinol) are pleiotropic regulatorycompounds that modulate the structure and function of a wide variety ofinflammatory, immune and structural cells. They are important regulatorsof epithelial cell proliferation, differentiation and morphogenesis inlungs. Retinoids exert their biological effects through a series ofhormone nuclear receptors that are ligand inducible transcriptionfactors belonging to the steroid/thyroid receptor super family.

The retinoid receptors are classified into two families, the retinoicacid receptors (RARs) and the retinoid X receptors (RXRs), eachconsisting of three distinct subtypes (α, β, and γ). Each subtype of theRAR gene family encodes a variable number of isoforms arising fromdifferential splicing of two primary RNA transcripts. All-trans retinoicacid is the physiological hormone for the retinoic acid receptors andbinds with approximately equal affinity to all the three RAR subtypes,but does not bind to the RXR receptors for which 9-cis retinoic acid isthe natural ligand. Retinoids have anti-inflammatory effects, alter theprogression of epithelial cell differentiation, and inhibit stromal cellmatrix production. These properties have led to the development oftopical and systemic retinoid therapeutics for dermatological disorderssuch as psoriasis, acne, and hypertrophic cutaneous scars. Otherapplications include the control of acute promyelocytic leukemia, adeno-and squamous cell carcinoma, and hepatic fibrosis.

A limitation in the therapeutic use of retinoids has stemmed from therelative toxicity observed with the naturally occurring retinoids,all-trans retinoic acid and 9-cis retinoic acid. These natural ligandsare non-selective in terms of RAR subtype and therefore have pleiotropiceffects throughout the body, which are often toxic.

Various retinoids have been described that interact selectively orspecifically with the RAR or RXR receptors or with specific subtypes (α,β, γ) within a class. RARA specific agonists have held high promise forthe treatment of cancers and many have entered human clinical trials.However, only one RARA specific agonist, tamibarotene, has ever beenapproved for the treatment of cancer. Moreover, tamibarotene is onlyapproved in Japan and only for the treatment of acute promyelocyticleukemia, despite trials in the US and Europe. The disconnect betweenthe theoretical efficacy of RARA agonists in cancer and the dearth ofregulatory approvals for such agents raises the question of why suchagonists are not effective and safe in humans. Therefore, there is aneed to better understand why RARA agonists have not met theirtherapeutic potential.

Recent advances in genomic technology and the understanding of generegulatory circuits has led to the discovery of super enhancers. Whereasmany genes in a given tissue or cancer type may be regulated by thepresence of enhancers in proximity to the gene coding region, a smallminority of these represent a highly asymmetric and disproportionatelylarge loading of transcriptional marks and machinery relative to allother active genes. Recent discoveries suggest that such enhancers aretied to genes of special relevance to the function and survival of thecell harboring them. As such, an association of a super enhancer with agene indicates the relative significance of said gene to the survival ofthat cell. These observations may be useful in predicting the efficacyof various therapies.

SUMMARY OF THE INVENTION

The present disclosure provides technologies for detecting one or moreof RARA super enhancer strength or ordinal rank, or RARA mRNA level thatis equal to or above a threshold value. The present disclosuredemonstrates that cells (e.g., cancer cells or cells from a subjectsuffering from MDS, e.g., AML cells, MDS cells, breast cancer cells)containing one or more of a RARA super enhancer strength or ordinal rankor a RARA mRNA level that is equal to or above a threshold value aremore sensitive to the anti-cancer effect of a RARA agonist (e.g., again-of-function RARA agonist; or a RARA-specific agonist (e.g., anagonist that is at least 10×, 100×, 1000×, 10000× or more specific forRAR-α, than for either of RAR-ß or RAR-γ)) than cells that are belowsuch threshold value.

The various embodiments, aspects and alternatives of this inventionsolve the problem of defining which cellular populations are sensitiveto agonists of retinoic acid receptor alpha (“RARA”), identifyingpatient subgroups that will benefit from treatment with RARA agonists(e.g., stratifying patients for treatment with a RARA agonist;separating RARA agonist responders from non-responders) and providingtreatment therapies directed at such patient subgroups. The solution isbased, at least in part, upon our discovery that a super-enhancer havinga strength or ordinal rank equal to or above a threshold value andassociated with a gene encoding retinoic acid receptor alpha (“RARA”) incertain cancer cells is indicative that such cell will respond totreatment with a RARA agonist. We have also discovered that RARA primaryRNA transcript levels, in particular mRNA levels, equal to or above athreshold level in certain cancer cells are also indicative that suchcancer cells will respond to treatment with a RARA agonist. In bothcases, the threshold values associated with these parameters appear tobe significantly more predictive than RARA protein levels.

In a first embodiment, the invention relates to a method of treating asubject suffering from cancer, wherein cancer cells in the subject havebeen determined to have one or more of:

a. a super enhancer associated with a RARA gene, wherein the superenhancer has a strength or ordinal rank that is equal to or above apre-determined threshold level; or

b. a level of primary RNA transcript from the RARA gene and/or a portionof the super enhancer associated therewith that is equal to or above apre-determined threshold level, wherein the method comprises the step ofadministering to the subject an agonist of RARA.

In some aspects of the first embodiment an agonist of RARA is onlyadministered if the super enhancer has a strength or ordinal rank thatis equal to or above a pre-determined threshold level; or the level ofprimary RNA transcript is equal to or above a pre-determined thresholdlevel.

In some aspects of the first embodiment, when the cancer cells in thesubject have been determined to have:

a. an enhancer or super enhancer associated with a RARA gene, whereinthe enhancer or super enhancer has a strength or ordinal rank that isbelow a pre-determined threshold level; or

b. a level of primary RNA transcript from the RARA gene and/or a portionof the super enhancer associated therewith that is below apre-determined threshold level, the method comprises the step ofadministering to the subject a therapeutic other than an agonist ofRARA.

In some aspects of the first embodiment, the cancer cells in the subjecthave been determined to have a super enhancer associated with a RARAgene that is at least 1.75-fold stronger than a portion of a superenhancer associated with MALAT1 as measured by ChIP-seq, wherein theportion is located at chr11:65263724-65266724 in genome build hg19, orat least an equivalent amount stronger than another reference enhanceror super enhancer locus.

In other aspects of the first embodiment, when the cancer cells in thesubject have been determined to meet either a. or b., the methodcomprises administering to the subject a therapeutic (e.g., atherapeutic agent or standard of care) other than an agonist of RARA,e.g., a chemotherapeutic agent or transplantation. In some embodiments,the subject has a cancer, e.g., a leukemia (e.g., acute myeloidleukemia). Exemplary therapeutics include chemotherapeutic agents (e.g.,cytarabine, gemcitabine, azacitidine, decitabine, fluorouracil, or ananthracycline (e.g., daunorubicin, doxorubicin, epirubicin, oridarubicin)) or transplantation (e.g., stem cell transplantation).

In alternate aspects of the first embodiment, the cancer cells in thesubject have been determined to have one of the following:

a. a super enhancer associated with a RARA gene that is at least1.5-fold higher in strength than a corresponding enhancer associatedwith a RARA gene in a human cell or human cell line known to benon-responsive to a RARA agonist; and/or

b. a RARA RNA primary transcript level that is at least 1.5-fold higherthan a corresponding RARA RNA primary transcript level in a human cellor human cell line known to be non-responsive to a RARA agonist.

In other alternate aspects of the first embodiment, the cancer cells inthe subject have been determined to have one the following:

a. a super enhancer associated with a RARA gene that has a strengthcorresponding to a prevalence rank that is equal to or above apre-determined prevalence cutoff for RARA gene super enhancer strength;and/or

b. a super enhancer associated with a RARA gene that has a strength thatis equal to or above a pre-determined RARA gene strength cutoff; and/or

c. a super enhancer associated with a RARA gene that has an ordinal ofstrength corresponding to a prevalence rank that is equal to or above apre-determined RARA gene strength ordinal prevalence cutoff; and/or

d. a super enhancer associated with a RARA gene that has an ordinal ofstrength that is equal to or above a pre-determined RARA gene strengthordinal cutoff; and/or

e. a RARA mRNA level that is equal to or above a pre-determined mRNAlevel cutoff; and or

f. a RARA mRNA level corresponding to a prevalence rank that is equal toor above a pre-determined RARA mRNA prevalence cutoff.

In certain more specific aspects, any of the pre-determined prevalencecutoffs set forth above is determined:

a. from a rank ordering of RARA super-enhancer strength in a populationof samples from the same type of cancer cells, wherein at least onesample has been determined to be responsive to the RARA agonist; and/or

b. from a rank ordering of RARA super-enhancer strength ordinal in apopulation of samples from the same type of cancer cells, wherein atleast one sample has been determined to be responsive to the RARAagonist; and/or

c. from a rank ordering of RARA mRNA levels in a population of samplesof the same type of cancer cells wherein at least one sample has beendetermined to be responsive to the RARA agonist.

In a second embodiment, the invention provides a method of treating asubject suffering from cancer comprising the steps of:

-   a. receiving information related to one or more of:    -   i. strength, ordinal rank or prevalence rank of a super enhancer        associated with a RARA gene in a cancer cell from the subject;        or    -   ii. level of primary RNA transcript from the RARA gene and/or a        portion of the super enhancer associated therewith in a cancer        cell from the subject; and-   b. administering to the subject an agonist of RARA if the    information indicates one or more of:    -   i. the super enhancer has a strength, ordinal rank, or        prevalence rank that is equal to or above a pre-determined        threshold level; or    -   ii. the level of RNA transcript is equal to or above a        pre-determined threshold level.

In one aspect of the second embodiment, the subject is administered atherapeutic other than an agonist of RARA if the information indicates:

-   -   iii. the super enhancer has a strength, ordinal rank, or        prevalence rank that is below a pre-determined threshold level;        or    -   iv. the level of RNA transcript is below a pre-determined        threshold level.

In one aspect of the second embodiment, step a. comprises receivinginformation related to one or more of:

-   -   i. the strength of a super enhancer associated with a RARA gene        compared to a control enhancer or super enhancer in the same        cancer; and/or    -   ii. the level of a RARA RNA primary transcript level compared to        a corresponding RARA RNA primary transcript level in a human        cell or human cell line known to be non-responsive to a RARA        agonist.

In one aspect of the second embodiment, step b comprises administeringto the subject an agonist of RARA if the information indicates one ormore of:

-   -   i. the super enhancer associated with a RARA gene is at least        1.75-fold stronger than a portion of a super enhancer associated        with MALAT1 located at chr11:65263724-65266724 in genome build        hg19, or at least an equivalent amount stronger than another        reference enhancer or super enhancer locus; and/or    -   ii. a RARA RNA primary transcript level that is at least        1.5-fold higher than a corresponding RARA RNA primary transcript        level in a human cell or human cell line known to be        non-responsive to a RARA agonist.

In an alternate aspect of the second embodiment, step a. comprisesreceiving information related to one or more of:

-   i. the strength of a super enhancer associated with a RARA gene    and/or the prevalence rank of RARA gene super enhancer strength in a    population to which the strength corresponds; and/or-   ii. the ordinal rank of the strength of a super enhancer associated    with a RARA gene as compared to other super enhancers in the cell    and/or the prevalence rank of a RARA gene super enhancer strength    ordinal in a population to which the ordinal rank corresponds;    and/or-   iii. RARA mRNA level and/or the prevalence rank of RARA mRNA level    in a population to which the mRNA level corresponds; and

step b. comprises administering to the subject an agonist of RARA if theinformation indicates one or more of:

-   -   i. the strength of a super enhancer associated with a RARA gene        is equal to or above a pre-determined cutoff value of RARA gene        super enhancer strength in a population; and/or    -   ii. the strength of a super enhancer associated with a RARA gene        corresponds to a prevalence rank that is equal to or above a        pre-determined prevalence cutoff of RARA gene super enhancer        strength in a population; and/or    -   iii. the ordinal rank of the strength of a super enhancer        associated with a RARA gene is equal to or above a        pre-determined ordinal cutoff value of RARA gene super enhancer        strength ordinal in a population; and/or    -   iv. the ordinal rank of strength of a super enhancer corresponds        to a prevalence rank that is equal to or above a pre-determined        prevalence cutoff of RARA gene super enhancer strength ordinal        in a population; and/or    -   v. the level of RARA mRNA is equal to or above a RARA mRNA level        that corresponds to a pre-determined cutoff value of RARA mRNA        level in a population; and/or    -   vi. the RARA mRNA level corresponds to a prevalence rank that is        equal to or above a pre-determined prevalence cutoff of RARA        mRNA level in a population.

In a third embodiment, the invention provides a packaged pharmaceuticalcomposition comprising:

-   a. a RARA agonist; and-   b. a written insert or label comprising instructions to use the RARA    agonist in a subject suffering from a cancer, and whose cancer cells    have been determined to have one or more of:    -   i. a super enhancer associated with a RARA gene, wherein the        super enhancer has a strength, ordinal rank or prevalence rank        that is equal to or above a pre-determined threshold level; or    -   ii. a level of primary RNA transcript from the RARA gene and/or        a portion of the super enhancer associated therewith that is        equal to or above a pre-determined threshold level.

In some aspects of the third embodiment, the written insert or labelcomprises instructions to use the RARA agonist in a subject whose cancercells that have been determined to have one or more of:

-   -   i. a super enhancer associated with a RARA gene that is at least        1.75-fold stronger than a portion of a super enhancer associated        with MALAT1 located at chr11:65263724-65266724 in genome build        hg19, or at least an equivalent amount stronger than another        reference enhancer or super enhancer locus; and/or    -   ii. a RARA RNA primary transcript level that is at least        1.5-fold higher than a corresponding RARA RNA primary transcript        level in a human cell or human cell line known to be        non-responsive to a RARA agonist.

In some aspects of the third embodiment, the written insert or labelcomprises instructions to use the RARA agonist in a subject whose cancercells that have been determined to have one or more of:

-   -   i. a super enhancer associated with a RARA gene that has a        strength that is equal to or above a pre-determined cutoff value        of RARA gene super enhancer strength in a population; and/or    -   ii. super enhancer associated with a RARA gene that has a        strength corresponding to a prevalence rank that is equal to or        above a pre-determined prevalence cutoff of RARA gene super        enhancer strength in a population; and/or    -   iii. a super enhancer associated with a RARA gene that has an        ordinal rank of the strength that is equal to or above a        pre-determined cutoff value of RARA gene super enhancer strength        ordinal in a population; and/or    -   iv. a super enhancer associated with a RARA gene that has an        ordinal rank of strength corresponding to a prevalence rank of        that is equal to or above a pre-determined RARA gene super        enhancer strength ordinal prevalence cutoff of in a population;        and/or    -   v. a level of RARA mRNA that is equal to or above a        pre-determined RARA mRNA level cutoff value in a population;        and/or    -   vi. a RARA mRNA level corresponding to a prevalence rank that is        equal to or above a pre-determined of RARA mRNA prevalence        cutoff in a population.

In a fourth embodiment, the invention provides a method of predictingthe efficacy of a RARA agonist in a treatment of a cancer in a subjectcomprising the step of determining if, having determined if, orreceiving information that:

-   -   a. a super enhancer associated with a RARA gene in the cancer        has a strength, ordinal rank or prevalence rank that is equal to        or above a pre-determined threshold level; or    -   b. a level of primary RNA transcript from the RARA gene and/or a        portion of the super enhancer associated therewith in the cancer        is equal to or above a pre-determined threshold level,        -   wherein any of a. or b. is predictive of RARA agonist            efficacy in the treatment.

In some aspects of the fourth embodiment predicting the efficacy of aRARA agonist in the treatment of the cancer in a subject comprises thestep of determining if the cancer is characterized by one or more of:

-   -   a. a super enhancer associated with a RARA gene is at least        1.75-fold stronger than a portion of a super enhancer associated        with MALAT1 located at chr11:65263724-65266724 in genome build        hg19, or at least an equivalent amount stronger than another        reference enhancer or super enhancer locus; and/or    -   b. a RARA RNA primary transcript level that is at least 1.5-fold        higher than a corresponding RARA RNA primary transcript level in        a human cell or human cell line known to be non-responsive to a        RARA agonist;        wherein any of a. or b. is predictive of RARA agonist efficacy        in the treatment.

In some aspects of the fourth embodiment predicting the efficacy of aRARA agonist in the treatment of the cancer in a subject comprises thestep of determining if the cancer is characterized by one or more of:

-   -   a. a super enhancer associated with a RARA gene that has a        strength that is equal to or above a pre-determined cutoff value        of RARA gene super enhancer strength in a population; and/or    -   b. a super enhancer associated with a RARA gene that has a        strength corresponding to a prevalence rank that is equal to or        above a pre-determined prevalence cutoff of RARA gene super        enhancer strength in a population; and/or    -   c. a super enhancer associated with a RARA gene that has an        ordinal rank of the strength of that is equal to or above a        pre-determined cutoff value of RARA gene super enhancer strength        ordinal in a population; and/or    -   d. a super enhancer associated with a RARA gene that has an        ordinal rank of strength corresponding to a prevalence rank of        that is equal to or above a pre-determined prevalence cutoff of        RARA gene super enhancer strength ordinal in a population;        and/or    -   e. a level of RARA mRNA that is equal to or above a        pre-determined cutoff value of RARA mRNA level in a population;        and/or    -   f. a level of RARA mRNA that corresponds to a prevalence rank        that is equal to or above a pre-determined prevalence cutoff of        RARA mRNA level in a population.

In a fifth embodiment, the invention provides a method of diagnosing,prognosing or treating a subject suffering from a cancer comprising thesteps of:

-   a. obtaining a sample of cancer cells from the subject;-   b. determining, having determined, or receiving information about    one or more of:    -   i. the strength, ordinal rank or prevalence rank of a super        enhancer associated with a RARA gene in the sample; or    -   ii. the level of primary RNA transcript from the RARA gene        and/or a portion of the super enhancer associated therewith in        the sample; and-   c. administering a therapeutic composition comprising a RARA agonist    if one or more of:    -   i. the super enhancer has a strength, ordinal rank or prevalence        rank that is equal to or above a pre-determined threshold level;        or    -   ii. the level of primary RNA transcript is equal to or above a        pre-determined threshold level.

In one aspect of the fifth embodiment, step b. comprises determining inthe sample one or more of:

-   -   i. the strength of a super enhancer associated with a RARA gene        compared to a control enhancer or super enhancer in the same        cancer; and/or    -   ii. the level of a RARA RNA primary transcript level compared to        a corresponding

RARA RNA primary transcript level in a human cell or human cell lineknown to be non-responsive to a RARA agonist.

In another aspect of the fifth embodiment, step c. comprisesadministering or recommending the administration of a therapeuticcomposition comprising a RARA agonist if one or more of:

-   -   i. the super enhancer associated with a RARA gene is at least        1.75-fold stronger than a portion of a super enhancer associated        with MALAT1 located at chr11:65263724-65266724 in genome build        hg19, or at least an equivalent amount stronger than another        reference enhancer or super enhancer locus;    -   ii. a RARA RNA primary transcript level that is at least        1.5-fold higher than a corresponding RARA RNA primary transcript        level in a human cell or human cell line known to be        non-responsive to a RARA agonist; and/or    -   iii. the RARA mRNA level is at least 1.5-fold higher than the        RARA mRNA level in a human cell or human cell line known to be        non-responsive to a RARA agonist.

In another aspect of the fifth embodiment, step b. comprises determiningin the sample one or more of:

-   -   i. strength of a super enhancer associated with a RARA gene        and/or the prevalence rank of strength of a super enhancer        associated with a RARA gene in a population to which the        strength corresponds; and/or    -   ii. ordinal rank of the strength of a super enhancer associated        with a RARA gene as compared to other super enhancers in the        cell and/or the prevalence rank of the ordinal rank of the        strength of a super enhancer associated with a RARA gene in a        population to which the ordinal rank corresponds; and/or    -   iii. RARA mRNA primary transcript level and/or the prevalence        rank of the RARA mRNA primary transcript in a population to        which the mRNA level corresponds.

In another aspect of the fifth embodiment, step c. comprisesadministering or recommending the administration of a therapeuticcomposition comprising a RARA agonist if one or more of:

-   -   a. a super enhancer associated with a RARA gene had a strength        that is equal to or above a pre-determined cutoff value of RARA        gene super enhancer strength in a population; and/or    -   b. a super enhancer associated with a RARA gene that has a        strength corresponding to a prevalence rank that is equal to or        above a pre-determined prevalence cutoff of RARA gene super        enhancer strength in a population; and/or    -   c. a super enhancer associated with a RARA gene that has an        ordinal rank of strength that is equal to or above a        pre-determined cutoff value of RARA gene super enhancer strength        ordinal in a population; and/or    -   d. a super enhancer associated with a RARA gene that has an        ordinal rank of strength corresponding to a prevalence rank that        is equal to or above a pre-determined prevalence cutoff of RARA        gene super enhancer strength ordinal in a population; and/or    -   e. a level of RARA mRNA is equal to or above a pre-determined        cutoff value of RARA mRNA level in a population; and/or    -   f. a level of RARA mRNA that corresponds to a prevalence rank        that is equal to or above a pre-determined prevalence cutoff of        RARA mRNA level in a population.

In a sixth embodiment, the invention provides a method of determiningthe level of RARA mRNA in a subject, comprising the steps of: a)obtaining total mRNA from a biological sample from the subject; b)appending to each mRNA molecule additional nucleotides not naturallyappended to such mRNA molecules to enable the mRNA molecules to bind toa solid support; c) sequencing the mRNA molecules; and d) determiningthe level of RARA mRNA.

In an alternate sixth embodiment the invention provides a method ofdetermining the level of RARA mRNA in a subject, comprising the stepsof: a) obtaining total mRNA from a biological sample from the subject;b) creating a cDNA library from the total mRNA; and c) combining thecDNA library with (i) a primer pair that is specific for cDNA createdfrom RARA mRNA; (ii) a DNA polymerase; and (iii) a component fordetection of any DNA molecules produced from the primer pair and the DNApolymerase. In some aspects of this alternate sixth embodiment, thecomponent for detection is a dye. In other aspects of this alternatesixth embodiment, the component for detection is a labelled (e.g.,radiolabelled or dye-labelled, oligonucleotide.

In some aspects of any of the sixth embodiments, the level of mRNAdetected in the subject is compared to a pre-determined threshold level.

In some aspects of any of the sixth embodiments, the patient issuffering from a cancer selected from AML, breast cancer or MDS. In morespecific aspects, the subject is suffering from one of the abovediseases and is administered a RARA agonist (e.g., tamibarotene) if thedetermined RARA mRNA level is equal to or above the pre-determinedthreshold.

In some aspects of any of the sixth embodiments, the pre-determinedthreshold is a RARA mRNA cutoff level determined by measuring RARA mRNAlevels in a population or population of samples having or representingthe same cancer; and identifying at least one population member that isresponsive to a RARA agonist. In some aspects of this sixth embodiment,the pre-determined threshold is a RARA mRNA cutoff level that isdetermined by calculating a prevalence cutoff based on RARAsuper-enhancer ordinal rank in a population or population of sampleswherein at least one population member is identified as being responsiveto a RARA agonist; and using the calculated prevalence cutoff todetermine a cutoff level of RARA mRNA levels in the same or a differentpopulation or population of samples.

In a seventh embodiment, the invention provides a composition comprising(i) cDNA reversed transcribed from mRNA obtained from a population ofcancer cells in a subject (e.g., bone marrow cells from a subjectsuffering from AML or MDS, breast cancer cells, etc.); (ii) a primerpair specific for cDNA transcribed from RARA mRNA; (iii) a DNApolymerase; and (iv) a component for detection of any DNA moleculesproduced from the primer pair and the DNA polymerase. In some aspects ofthis seventh embodiment, the component for detection is a dye. In otheraspects of this seventh embodiment, the component for detection is alabelled (e.g., radiolabelled or dye-labelled, oligonucleotide. In someaspects of this seventh embodiment, the composition is used to determineRARA mRNA level in the subject. In more specific aspects of this seventhembodiment, the determined RARA mRNA levels are compared to a cutoffvalue and the comparison is utilized to determine whether the patientshould be administered a RARA agonist (e.g., tamibarotene).

In an eighth embodiment, the invention provides a differential method oftreating a set of subjects suffering from cancer comprisingadministering a RARA agonist to a subset of subjects whose cancer ischaracterized by a RARA mRNA level that is equal to or above apre-determined threshold; and not administering a RARA agonist to asubset of subjects whose cancer is characterized by a RARA mRNA levelthat is below a pre-determined threshold.

In some aspects of this eighth embodiment, the set of subjects issuffering from the same cancer and the cancer is selected from AML,breast cancer or MDS. In some aspects of the seventh embodiment, theRARA agonist is tamibarotene. In some aspects of this seventhembodiment, the pre-determined threshold is an mRNA cutoff leveldetermined by measuring RARA mRNA levels in a population or populationof samples having or representing the same cancer; and identifying atleast one population member that is responsive to a RARA agonist. Insome aspects of this seventh embodiment, the pre-determined threshold isa mRNA cutoff level determined by calculating a prevalence cutoff basedon RARA super-enhancer ordinal rank in a population or population ofsamples wherein at least one population member is identified as beingresponsive to a RARA agonist; and using the calculated prevalence cutoffto determine a cutoff level of RARA mRNA levels in the same or adifferent population or population of samples.

In a ninth embodiment, the invention provides a method comprising thesteps of:

-   -   a. obtaining cancer cells from a subject suffering from cancer;        and    -   b. measuring in the cancer cells:        -   i. the strength, ordinal rank or prevalence rank of a super            enhancer associated with a RARA gene in the sample; or        -   ii. the level of primary RNA transcript from the RARA gene            and/or a portion of the super enhancer associated therewith            in the sample; and    -   c. comparing the measurement obtained in step b. to a threshold        value.

In any and all embodiments, in some aspects, the present inventionfeatures a pharmaceutical composition for use in treating a subjectsuffering from cancer, wherein the composition comprises an agonist ofRARA.

The details of one or more embodiments of the invention are set forthherein. Other features, objects, and advantages of the invention will beapparent from the Detailed Description, the Figures, the Examples, andthe Embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B graphically depict the loge enrichment of RARA superenhancer as measured by ChIP-qPCR.

FIG. 2 depicts the level of H3K27Ac reads across the RARA locus asdetermined by ChIP-seq in 5 different breast cancer cell lines. “RARA”indicates the location of the RARA gene in the fragment of DNA analyzed.

FIGS. 3A-3M are listings of ChIP-seq results for a wide variety of celllines and patient samples. It shows the relative strength of the RARAsuper enhancer as compared to a portion of the MALAT1 super enhancer ineach of these cells or patient samples.

FIG. 4A shows the rank ordering of RARA SE strength, expressed as log₁₀RARA/MALAT1 for all breast cancer cell lines and patient samplesanalyzed by ChIP-seq in FIG. 3 . FIG. 4B shows the rank ordering of RARASE strength, expressed as log₁₀ RARA/MALAT1 for all AML cancer celllines and patient samples analyzed by ChIP-seq in FIG. 3 .

FIG. 5 shows the rank ordering of RARA SE strength, expressed as log₁₀RARA/MALAT1 for all normal hematological cell lines and patient samplesanalyzed by ChIP-seq in FIG. 3 .

FIG. 6 shows a scatter plot of RARA SE strength, expressed as log₁₀RARA/MALAT1 for patient samples versus cell lines in the various AML andbreast cancer cells analyzed by ChIP-seq in FIG. 3 .

FIG. 7 shows the viability of various breast cancer cell lines in thepresence of varying doses of tamibarotene after 5 days of treatment.

FIG. 8 shows the correlation between the log₁₀ of tamibarotene EC₅₀versus RARA SE strength (RARA/MALAT1 fold enrichment) for sevendifferent breast cell cancer lines.

FIG. 9 shows the viability of various AML cell lines in the presence ofvarying doses of tamibarotene after 5 days of treatment.

FIG. 10 shows the correlation between the log₁₀ of tamibarotene EC₅₀versus for eleven different AML cell lines.

FIG. 11 shows the mRNA expression levels of the three different RARsubtypes (RaR-α (“RARA”); RaR-ß (“RARB”); and RaR-γ (“RARG”)), asmeasured by Affymetrix Array-Based analysis for a tamibaroteneresponsive (Au565) and non-responsive (HCC1143) breast cancer cell line.

FIG. 12 show the correlation between mRNA expression (log₂(1+FPKM)) andRARA SE strength (RARA/MALAT1 fold enrichment) for 48 different AMLpatient samples using RNA-Seq.

FIG. 13 shows the inverse mRNA expression level as measured by rt-qPCRand expressed as dCt for 5 different breast cancer cell lines.

FIG. 14A shows the correlation between mRNA expression levels asmeasured by rt-qPCR and RARA SE strength (RARA/MALAT1 fold enrichment)for seven different breast cell cancer lines. FIG. 14B shows thecorrelation between mRNA expression levels as measured by rt-qPCR andresponsiveness to tamibarotene (as measured by log₁₀ EC₅₀) for sevendifferent breast cell cancer lines.

FIG. 15 is a Western blot depicting the protein level of three differentknown RARA isoforms in 5 different breast cell cancer lines.

FIGS. 16A-16B depict the HER2 and RARA gene copy numbers in atamibarotene weakly responsive (T47D, FIG. 16A) and a highly responsive(AU565, FIG. 16B) breast cancer cell line.

FIGS. 17A-17C depict the level of H3K27Ac reads across the RARA locus asdetermined by ChIP-seq for glioblastoma (FIG. 17A), neuroblastoma (FIG.17B), and colorectal (FIG. 17C) cancer patient samples.

FIG. 18 depicts the response to various daily dosages of tamibarotene ina breast cancer cell line (HCC1945)-derived mouse xenograft model ofbreast cancer.

FIG. 19 depicts a log₁₀ rank-ordered graph of RARA super enhancerstrength ordinal in 80 breast cancer samples including the breast cancercell line HCC1945.

FIG. 20A depicts a log₁₀ rank-ordered graph of RARA super enhancerstrength ordinal in 48 patient breast cancer samples. The lightercolored bars represent samples whose RARA super enhancer strengthordinal was equal to or above the prevalence cutoff. Darker colored barsrepresent samples whose RARA super enhancer strength ordinal was belowthe prevalence cutoff. FIG. 20B depicts the same rank ordered graph asPanel A and further indicates the specific subtype of breast cancer(hormone receptor positive (HR⁺), HER2 positive (HER2⁺), or triplenegative (TN)), as well as the calculated prevalence cutoff for eachsubtype.

FIGS. 21A-B depict the response to daily dosing of tamibarotene (SY1425)in two different patient sample-derived mouse xenograft breast cancermodels.

FIG. 22 depicts that RARA mRNA levels in nine different patientsample-derived mouse xenograft breast cancer models. The white barrepresents both the value for CTG-1124, as well as the 55.3% prevalencecutoff in this population.

FIG. 23A depicts RARA super enhancer strength in 6 AML cell lines. FIG.23B depicts the responsiveness of those same 6 AML cell lines totamibarotene treatment. FIG. 23C depicts a log₁₀ rank-ordered graph ofRARA super enhancer strength ordinal in 94 AML samples including four ofthe six AML cell lines analyzed in FIGS. 20A and 20B—Sig-M5, MV411, HELand Kasumi.

FIG. 24 depicts a log₁₀ rank-ordered graph of RARA super enhancerstrength ordinal in 70 AML patient samples. The lighter colored barsrepresent samples whose RARA super enhancer strength ordinal was equalto or above the prevalence cutoff. Darker colored bars represent sampleswhose RARA super enhancer strength ordinal was below the prevalencecutoff.

FIG. 25 depicts RARA mRNA levels in the 70 AML patient samples used inFIG. 24 and binned according to whether their RARA super enhancerstrength ordinal was above (or equal to) the prevalence cutoff (“HighRARA”) or below the prevalence cutoff (“Low RARA”).

FIGS. 26A and 26D depict the response, as measured by % CD45⁺ cells, todaily dosing of tamibarotene in two different patient-derived mousexenograft AML models. FIGS. 26B and 26E depict the % CD45⁺ cells indifferent organs and biological fluids, and FIGS. 26C and 26F show thetime of survival of the mouse models.

FIG. 27A-B depict the response, as measured by % CD45⁺ cells, to dailydosing of tamibarotene in two additional patient-derived mouse xenograftAML models. FIG. 27C depicts the % CD45⁺ cells in different organs andbiological fluids in one of those models, and FIG. 27D depicts the timeof survival in that model.

FIG. 28 depicts the RARA mRNA levels in 64 AML patient samples,including the four used to create mouse xenograft models (see FIGS.26A-26F and FIGS. 27A-27D). The lighter colored bars represent sampleswhose RARA mRNA was equal to or above the prevalence cutoff asdetermined from RARA super enhancer strength ordinal. Darker coloredbars represent samples whose RARA mRNA level was below that prevalencecutoff.

FIG. 29A depicts the response of AM5512 xenograft mice to 4 mg/kg ATRABID, 3 mg/kg tamibarotene BID and a vehicle control as measured by %CD45⁺ cells. FIG. 29B depicts the survival rate of such mice during thecourse of the experiment.

FIG. 30 is a table of different cancer types where greater than 5% ofthe samples tested had RARA mRNA levels at least 2 standard deviationsabove the mean.

FIG. 31 depicts RARA mRNA levels in MDS patient samples versus cellsfrom healthy (“Normal”) patients.

FIG. 32 depicts the correlation between RARA enhancer strength andsensitivity to tamibarotene in 11 different AML cell lines.

FIG. 33 depicts the correlation between RARA mRNA level and sensitivityto tamibarotene in 11 different AML cell lines.

DEFINITIONS

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and lower animalswithout undue toxicity, irritation, allergic response and the like, andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, Berge et al.,describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein byreference. Pharmaceutically acceptable salts of the compounds of thisinvention include those derived from suitable inorganic and organicacids and bases. Examples of pharmaceutically acceptable, nontoxic acidaddition salts are salts of an amino group formed with inorganic acidssuch as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuricacid, and perchloric acid or with organic acids such as acetic acid,oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, ormalonic acid or by using other methods known in the art such as ionexchange. Other pharmaceutically acceptable salts include adipate,alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate,borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, MALAT1e, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄ alkyl)₄ ⁻ salts.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, lower alkyl sulfonate, and aryl sulfonate.

The term “solvate” refers to forms of the compound that are associatedwith a solvent, usually by a solvolysis reaction. This physicalassociation may include hydrogen bonding. Conventional solvents includewater, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and thelike. The compounds described herein, such as of Formula (I) may beprepared, e.g., in crystalline form, and may be solvated. Suitablesolvates include pharmaceutically acceptable solvates and furtherinclude both stoichiometric solvates and non-stoichiometric solvates. Incertain instances, the solvate will be capable of isolation, forexample, when one or more solvent molecules are incorporated in thecrystal lattice of a crystalline solid. “Solvate” encompasses bothsolution-phase and isolable solvates. Representative solvates includehydrates, ethanolates, and methanolates.

The term “hydrate” refers to a compound which is associated with water.Typically, the number of the water molecules contained in a hydrate of acompound is in a definite ratio to the number of the compound moleculesin the hydrate. Therefore, a hydrate of a compound may be represented,for example, by the general formula R·x H2O, wherein R is the compoundand wherein x is a number greater than 0. A given compound may form morethan one type of hydrates, including, e.g., monohydrates (x is 1), lowerhydrates (x is a number greater than 0 and smaller than 1, e.g.,hemihydrates (R·0.5 H₂O)), and polyhydrates (x is a number greater than1, e.g., dihydrates (R·2 H₂O) and hexahydrates (R·6 H₂O)).

A “subject” to which administration is contemplated includes, but is notlimited to, humans (i.e., a male or female of any age group, e.g., apediatric subject (e.g., infant, child, adolescent) or adult subject(e.g., young adult, middle-aged adult, or senior adult)) and/or othernon-human animals, for example, mammals (e.g., primates (e.g.,cynomolgus monkeys, rhesus monkeys); commercially relevant mammals suchas cattle, pigs, horses, sheep, goats, cats, and/or dogs) and birds(e.g., commercially relevant birds such as chickens, ducks, geese,and/or turkeys). In certain embodiments, the animal is a mammal. Theanimal may be a male or female and at any stage of development. Anon-human animal may be a transgenic animal. In certain embodiments, thesubject is a human.

The terms “administer,” “administering,” or “administration,” as usedherein refers to implanting, absorbing, ingesting, injecting, inhaling,or otherwise introducing an inventive compound, or a pharmaceuticalcomposition thereof.

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a “pathological condition” (e.g., a disease, disorder, orcondition, or one or more signs or symptoms thereof) described herein.In some embodiments, “treatment,” “treat,” and “treating” require thatsigns or symptoms of the disease disorder or condition have developed orhave been observed. In other embodiments, treatment may be administeredin the absence of signs or symptoms of the disease or condition. Forexample, treatment may be administered to a susceptible individual priorto the onset of symptoms (e.g., in light of a history of symptoms and/orin light of genetic or other susceptibility factors). Treatment may alsobe continued after symptoms have resolved, for example, to delay orprevent recurrence.

As used herein, the terms “condition,” “disease,” and “disorder” areused interchangeably.

An “effective amount” of a compound described herein, such as of Formula(I) refers to an amount sufficient to elicit the desired biologicalresponse, i.e., treating the condition. As will be appreciated by thoseof ordinary skill in this art, the effective amount of a compounddescribed herein, such as of Formula (I) may vary depending on suchfactors as the desired biological endpoint, the pharmacokinetics of thecompound, the condition being treated, the mode of administration, andthe age and health of the subject. An effective amount encompassestherapeutic and prophylactic treatment. For example, in treating cancer,an effective amount of an inventive compound may reduce the tumor burdenor stop the growth or spread of a tumor.

A “therapeutically effective amount” of a compound described herein,such as of Formula (I) is an amount sufficient to provide a therapeuticbenefit in the treatment of a condition or to delay or minimize one ormore symptoms associated with the condition. In some embodiments, atherapeutically effective amount is an amount sufficient to provide atherapeutic benefit in the treatment of a condition or to minimize oneor more symptoms associated with the condition. A therapeuticallyeffective amount of a compound means an amount of therapeutic agent,alone or in combination with other therapies, which provides atherapeutic benefit in the treatment of the condition. The term“therapeutically effective amount” can encompass an amount that improvesoverall therapy, reduces or avoids symptoms or causes of the condition,or enhances the therapeutic efficacy of another therapeutic agent.

The terms “neoplasm” and “tumor” are used herein interchangeably andrefer to an abnormal mass of tissue wherein the growth of the masssurpasses and is not coordinated with the growth of a normal tissue. Aneoplasm or tumor may be “benign” or “malignant,” depending on thefollowing characteristics: degree of cellular differentiation (includingmorphology and functionality), rate of growth, local invasion, andmetastasis. A “benign neoplasm” is generally well differentiated, hascharacteristically slower growth than a malignant neoplasm, and remainslocalized to the site of origin. In addition, a benign neoplasm does nothave the capacity to infiltrate, invade, or metastasize to distantsites. Exemplary benign neoplasms include, but are not limited to,lipoma, chondroma, adenomas, acrochordon, senile angiomas, seborrheickeratoses, lentigos, and sebaceous hyperplasias. In some cases, certain“benign” tumors may later give rise to malignant neoplasms, which mayresult from additional genetic changes in a subpopulation of the tumor'sneoplastic cells, and these tumors are referred to as “pre-malignantneoplasms.” An exemplary pre-malignant neoplasm is a teratoma. Incontrast, a “malignant neoplasm” is generally poorly differentiated(anaplasia) and has characteristically rapid growth accompanied byprogressive infiltration, invasion, and destruction of the surroundingtissue. Furthermore, a malignant neoplasm generally has the capacity tometastasize to distant sites.

As used herein, the term “cancer” refers to a malignant neoplasm(Stedman's Medical Dictionary, 25th ed.; Hensly ed.; Williams & Wilkins:Philadelphia, 1990). Exemplary cancers include, but are not limited to,acoustic neuroma; adenocarcinoma; adrenal gland cancer; anal cancer;angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma,hemangiosarcoma); appendix cancer; benign monoclonal gammopathy; biliarycancer (e.g., cholangiocarcinoma); bladder cancer; breast cancer (e.g.,adenocarcinoma of the breast, papillary carcinoma of the breast, mammarycancer, medullary carcinoma of the breast); brain cancer (e.g.,meningioma, glioblastomas, glioma (e.g., astrocytoma,oligodendroglioma), medulloblastoma); bronchus cancer; carcinoid tumor;cervical cancer (e.g., cervical adenocarcinoma); choriocarcinoma;chordoma; craniopharyngioma; connective tissue cancer; epithelialcarcinoma; ependymoma; endotheliosarcoma (e.g., Kaposi's sarcoma,multiple idiopathic hemorrhagic sarcoma); endometrial cancer (e.g.,uterine cancer, uterine sarcoma); esophageal cancer (e.g.,adenocarcinoma of the esophagus, Barrett's adenocarcinoma); Ewing'ssarcoma; eye cancer (e.g., intraocular melanoma, retinoblastoma);familiar hypereosinophilia; gall bladder cancer; gastric cancer (e.g.,stomach adenocarcinoma); gastrointestinal stromal tumor (GIST); germcell cancer; head and neck cancer (e.g., head and neck squamous cellcarcinoma, oral cancer (e.g., oral squamous cell carcinoma), throatcancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngealcancer, oropharyngeal cancer)); hematopoietic cancers (e.g., leukemiasuch as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL),acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronicmyelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chroniclymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL)); lymphomasuch as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) andnon-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large celllymphoma (DLCL) (e.g., diffuse large B-cell lymphoma), follicularlymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas(e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma),primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacyticlymphoma (i.e., Waldenstrom's macroglobulinemia), hairy cell leukemia(HCL), immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma and primary central nervous system (CNS) lymphoma; and T-cellNHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheralT-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g.,mycosis fungoides, Sezary syndrome), angioimmunoblastic T-cell lymphoma,extranodal natural killer T-cell lymphoma, enteropathy type T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, and anaplasticlarge cell lymphoma); a mixture of one or more leukemia/lymphoma asdescribed above; and multiple myeloma (MM)), heavy chain disease (e.g.,alpha chain disease, gamma chain disease, mu chain disease);hemangioblastoma; hypopharynx cancer; inflammatory myofibroblastictumors; immunocytic amyloidosis; kidney cancer (e.g., nephroblastomaa.k.a. Wilms' tumor, renal cell carcinoma); liver cancer (e.g.,hepatocellular cancer (HCC), malignant hepatoma); lung cancer (e.g.,bronchogenic carcinoma, small cell lung cancer (SCLC), non-small celllung cancer (NSCLC), adenocarcinoma of the lung); leiomyosarcoma (LMS);mastocytosis (e.g., systemic mastocytosis); muscle cancer;myelodysplastic syndrome (MDS); mesothelioma; myeloproliferativedisorder (MPD) (e.g., polycythemia vera (PV), essential thrombocytosis(ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF),chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML),chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES));neuroblastoma; neurofibroma (e.g., neurofibromatosis (NF) type 1 or type2, schwannomatosis); neuroendocrine cancer (e.g., gastroenteropancreaticneuroendocrine tumor (GEP-NET), carcinoid tumor); osteosarcoma (e.g.,bone cancer); ovarian cancer (e.g., cystadenocarcinoma, ovarianembryonal carcinoma, ovarian adenocarcinoma); papillary adenocarcinoma;pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductalpapillary mucinous neoplasm (IPMN), Islet cell tumors); penile cancer(e.g., Paget's disease of the penis and scrotum); pinealoma; primitiveneuroectodermal tumor (PNT); plasma cell neoplasia; paraneoplasticsyndromes; intraepithelial neoplasms; prostate cancer (e.g., prostateadenocarcinoma); rectal cancer; rhabdomyosarcoma; salivary gland cancer;skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA),melanoma, basal cell carcinoma (BCC)); small bowel cancer (e.g.,appendix cancer); soft tissue sarcoma (e.g., malignant fibroushistiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor(MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma); sebaceous glandcarcinoma; small intestine cancer; sweat gland carcinoma; synovioma;testicular cancer (e.g., seminoma, testicular embryonal carcinoma);thyroid cancer (e.g., papillary carcinoma of the thyroid, papillarythyroid carcinoma (PTC), medullary thyroid cancer); urethral cancer;vaginal cancer; and vulvar cancer (e.g., Paget's disease of the vulva).

The term “biological sample” refers to any sample including tissuesamples (such as tissue sections and needle biopsies of a tissue); cellsamples (e.g., cytological smears (such as Pap or blood smears) orsamples of cells obtained by microdissection); samples of wholeorganisms (such as samples of yeasts or bacteria); or cell fractions,fragments or organelles (such as obtained by lysing cells and separatingthe components thereof by centrifugation or otherwise). Other examplesof biological samples include blood, serum, urine, semen, fecal matter,cerebrospinal fluid, interstitial fluid, mucus, tears, sweat, pus,biopsied tissue (e.g., obtained by a surgical biopsy or needle biopsy),nipple aspirates, milk, vaginal fluid, saliva, swabs (such as buccalswabs), or any material containing biomolecules that is derived from afirst biological sample. Biological samples also include thosebiological samples that are transgenic, such as transgenic oocyte, spermcell, blastocyst, embryo, fetus, donor cell, or cell nucleus. In someaspect, a biological sample from a subject suffering from AML or MDS isa bone marrow aspirate.

The term “RARA gene” refers to a genomic DNA sequence that encodes afunctional retinoic acid receptor-α and specifically excludes genefusions that comprise all or a portion of the RARA gene. In someembodiments, the RARA gene is located at chr17:38458152-38516681ingenome build hg19.

The term “enhancer” refers to a region of genomic DNA acting to regulategenes up to 1 Mbp away. An enhancer may overlap, but is often notcomposed of, gene coding regions. An enhancer is often bound bytranscription factors and designated by specific histone marks.

The term “super enhancer” refers to a subset of enhancers that contain adisproportionate share of histone marks and/or transcriptional proteinsrelative to other enhancers in a particular cell. Because of this, agene regulated by a super enhancer is predicted to be of high importanceto the function of that cell. Super enhancers are typically determinedby rank ordering all of the enhancers in a cell based on strength anddetermining using available software such as ROSE (available on theworld-wide web at bitbucket.org/young computation/rose), the subset ofenhancers that have significantly higher strength than the medianenhancer in the cell (see, e.g., U.S. Pat. No. 9,181,580, which isherein incorporated by reference).

The term “primary RNA transcript” as used herein refers to the RNAtranscription product from the DNA sequence that include one or more ofthe gene coding region, and an enhancer, or a super enhancer associatedwith that gene. In some embodiments, the term “primary RNA transcript”is interchangeable with the term “eRNA” or “enhancer RNA” when such RNAincludes RNA derived from the DNA corresponding to the enhancer region.In other embodiments, the term “primary RNA transcript” refers to themRNA transcribed from the gene coding region.

The term “strength” when referring to a portion of an enhancer or asuper enhancer, as used herein means the area under the curve of thenumber of H3K27Ac or other genomic marker reads plotted against thelength of the genomic DNA segment analyzed. Thus, “strength” is anintegration of the signal resulting from measuring the mark at a givenbase pair over the span of the base pairs defining the region beingchosen to measure.

The term “prevalence rank” for a specified value (e.g., the strength ofa super enhancer associated with a RARA gene) means the percentage of apopulation that are equal to or greater than that specific value. Forexample a 35% prevalence rank for the strength of a super enhancerassociated with a RARA gene in a test cell means that 35% of thepopulation have a RARA gene enhancer with a strength equal to or greaterthan the test cell.

The term “prevalence cutoff” for a specified value (e.g., the strengthof a super enhancer associated with a RARA gene) means the prevalencerank that defines the dividing line between two subsets of a population(e.g., responders and non-responders). Thus, a prevalence rank that isequal to or higher (i.e., a lower percentage value) than the prevalencecutoff defines one subset of the population; and a prevalence rank thatis lower (e.g., a higher percentage value) than the prevalence cutoffdefines the other subset of the population.

The terms “cutoff” and “cutoff value” mean a value measured in an assaythat defines the dividing line between two subsets of a population(e.g., responders and non-responders). Thus, a value that is equal to orhigher than the cutoff value defines one subset of the population; and avalue that is lower than the cutoff value defines the other subset ofthe population.

The terms “threshold” and “threshold level” mean a level that definesthe dividing line between two subsets of a population (e.g., respondersand non-responders). A threshold level may be a prevalence cutoff or acutoff value.

The term “population” or “population of samples” means a sufficientnumber (e.g., at least 30, 40, 50 or more) of different samples thatreasonably reflects the distribution of the value being measured in alarger group. Each sample in a population of samples may be a cell line,a biological sample obtained from a living being (e.g., a biopsy orbodily fluid sample), or a sample obtained from a xenograft (e.g., atumor grown in a mouse by implanting a cell line or a patient sample),wherein each sample is from a living being suffering from or from a cellline or xenograft representing, the same disease, condition or disorder.

The term “ordinal rank” of a specified value means the rank order ofthat value as compared to a set of other values. For example, an ordinalrank of 100 in terms of the strength of a super enhancer associated witha RARA gene in a test cell as compared to other super enhancers in thetest cell means that 99 other super enhancers in the test cell hadgreater strength than the super enhancer associated with a RARA gene.

The term “rank ordering” means the ordering of values from highest tolowest or from lowest to highest.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION RARASuper-Enhancer Identification and Determination of Threshold Levels

The identification of an enhancer or super enhancer may be achieved byvarious methods known in the art, for example as described in Cell 2013,155, 934-947 and PCT/US2013/066957 (the international correlate of U.S.application Ser. No. 14/523,560, now U.S. Pat. No. 9,181,580), both ofwhich are incorporated herein by reference. In some embodiments, theidentification of a super enhancer is achieved by obtaining cellularmaterial and DNA from a cancer sample in a patient (e.g., from abiopsy). The important metrics for enhancer measurement occur in twodimensions—the length of the DNA over which genomic markers (e.g.,H3K27Ac) are contiguously detected—and the compiled incidence of genomicmarker at each base pair along that span of DNA constituting themagnitude. The measurement of the area under the curve (“AUC”) resultingfrom integration of length and magnitude analysis determines thestrength of the enhancer. It is the strength of the RARA super enhancerrelative to a control that is used in one aspect of the presentinvention to determine whether or not a subject will be responsive aRARA agonist. It will be readily apparent to those of skill in the artthat if the length of DNA over which the genomic markers is detected isthe same for both RARA and the control, then the ratio of the magnitudeof the RARA super enhancer relative to the control will be equivalent tothe strength and may also be used to determine whether or not a subjectwill be responsive a RARA agonist.

We have determined through H3K27Ac ChIP-seq methods that there is asuper-enhancer locus associated with the RARA gene atchr17:38458152-38516681 (genome build hg19). This locus actuallyoverlaps the RARA gene locus itself and therefore was considered to be asuper-enhancer locus associated with that gene because ofproximity/overlap. Thus, in some embodiments, determination of thestrength of a super-enhancer associated with the RARA gene according tothe present invention only requires analysis of this specific portion ofthe genome, as opposed to requiring an analysis of the entire genome.

ChIP-sequencing, also known as ChIP-seq, is used to analyze proteininteractions with DNA. ChIP-seq combines chromatin immunoprecipitation(ChIP) with massively parallel DNA sequencing to identify the bindingsites of DNA-associated proteins. It can be used to map global bindingsites precisely for any protein of interest. Previously, ChIP-on-chipwas the most common technique utilized to study these protein-DNArelations. Successful ChIP-seq is dependent on many factors includingsonication strength and method, buffer compositions, antibody quality,and cell number.; see, e.g., T. Furey, Nature Reviews Genetics 13,840-852 (December 2012); M. L. Metzker, Nature Reviews Genetics 11,31-46 (January 2010); and P. J. Park, Nature Reviews Genetics 10,669-680 (October 2009)) . Genomic markers other that H3K27Ac that can beused to identify super enhancers using ChIP-seq include, P300, CBP,BRD2, BRD3, BRD4, components of the mediator complex (J Loven, et al.,Cell, 153(2):320-334, 2013), histone 3 lysine 4 monomethylated(H3K4me1), or other tissue specific enhancer tied transcription factors(E Smith & A Shilatifard, Nat Struct Mol Biol, 21(3):210-219, 2014) (SPott & Jason Lieb, Nature Genetics, 47(1):8-12, 2015).

In some embodiments, H3K27ac or other marker ChIP-seq datasuper-enhancer maps of the entire genome of a cell line or a patientsample already exist. In these embodiments, one would simply determinewhether the strength, or ordinal rank of the enhancer or super-enhancerin such maps at the chr17:38458152-38516681 (genome build hg19) locuswas equal to or above the pre-determined threshold level.

In some embodiments, determination of whether or not the strength of theenhancer or super-enhancer at the chr17:38458152-38516681 locus requiresa comparison of the ChIP-seq reads in this region to a region known tocomprise a ubiquitous super-enhancer or enhancer that is present atsimilar levels in all cells. One example of such a ubiquitoussuper-enhancer region is the MALAT1 super-enhancer locus(chr11:65263724-65266724). By comparing the ChIP-seq reads at the RARAlocus with that at the MALAT1 locus, one can determine whether or notthe strength of a super-enhancer at the RARA locus is equal to or abovethe predetermined threshold level and whether or not the cells thereinwill respond to a RARA agonist.

In some embodiments of the present invention, the threshold level iswhen log₁₀ (AUC of ChIP-seq reads at the RARA locus (“R”)/AUC ofChIP-seq reads at the MALAT1 super-enhancer locus (“M”)) is 0.25 orgreater. In some aspects of these embodiments, the threshold level foridentifying responders to a RARA agonist is log₁₀ (R/M) of 0.3 orgreater, 0.35 or greater, or 0.4 or greater.

In some embodiments of the present invention, the threshold level iswhen (AUC of ChIP-seq reads at the RARA locus (“R”)/AUC of ChIP-seqreads at the MALAT1 super-enhancer locus (“M”)) is 1.75 or greater. Insome aspects of these embodiments, the threshold level for identifyingresponders to a RARA agonist is log₁₀ (R/M) of 2.0 or greater, 2.25 orgreater, or 2.75 or greater.

In some embodiments of the present invention R, as defined above, iscompared to a control enhancer or super enhancer locus other than MALAT1(the number of ChIP-seq reads at this other control enhancer or superenhancer is referred to as “C”). When another control enhancer or superenhancer locus, C, is utilized, the threshold values expressed as log₁₀(“V”), referred to above for comparison to M, e.g., log₁₀(R/M)≥0.25,log₁₀(R/M)≥0.3, log₁₀(R/M)≥0.35, or log₁₀(R/M)≥0.4, must be adjusted toan equivalent value to compare to C in order to account for the relativestrength of C as compared to M. This “equivalent adjusted thresholdvalue” (“A”) is calculated as follows:

A=log₁₀(M/C)+V

As a non-limiting example, if the calculated strength of the MALAT1super enhancer (M) is 10-fold greater than the control enhancer or superenhancer used as a comparator (C), and the threshold value (V) is 0.25,then A=log₁₀(10) +0.25=1.25 and the adjusted threshold value is 1.25.For this example, when C is used as the comparator, then log₁₀(R/C)equal or greater than 1.25 is considered the equivalent to a log₁₀(R/M)equal to or greater than 0.25 when M is used as the comparator. It willbe readily apparent that an adjusted threshold value can be calculatedin a similar manner for any additional comparator based on its relativestrength to either MALAT1 or any other comparator for which an adjustedthreshold value has already been determined.

The same adjustments above can be made when linear values compared to Mare used as threshold levels (e.g., ≥1.75-fold, ≥2.0-fold, ≥2.25-fold,or 2.5-fold). In this case, one obtains the ratio of M to C, and thenmultiplies the threshold value by that ratio to obtain appropriatethreshold values when comparing R to C (i.e., (thresholdvalue)c=(M/C)(threshold value)M)

It should be understood that the specific chromosomal location of bothRARA and MALAT1 may differ for different genome builds and/or fordifferent cell types. However, one of skill in the art can determinesuch different locations by locating in such other genome builds,specific sequences corresponding to the RARA and/or MALAT1 loci ingenome build hg 19.

Other methods for identifying super enhancers include chromatinimmunoprecipitation (J E Delmore, et al., Cell, 146(6)904-917, 2011) andchip array (ChIP-chip), and chromatin immunoprecipitation followed byqPCR (ChIP-qPCR) using the same immunoprecipitated genomic markers andoligonucleotide sequences that hybridize to the chr17:38458152-38516681(genome build hg19) RARA locus. In the case of ChIP-chip, the signal istypically detected by intensity fluorescence resulting fromhybridization of a probe and input assay sample as with other arraybased technologies. For ChIP-qPCR, a dye that becomes fluorescent onlyafter intercalating the double stranded DNA generated in the PCRreaction is used to measure amplification of the template.

In some embodiments, determination of whether a cell has a RARA superenhancer above a requisite threshold level is achieved by comparing RARAenhancer strength in a test cell to the corresponding RARA strength in acell known to not respond to RARA (a “control cell”). Preferably thecontrol cell is the same cell type as the test cell. In one aspect ofthese embodiments, the control cell is such cell in a HCC1143. Inanother aspect of these embodiments, the control cell is any cell listedin FIGS. 3A-3M, wherein log₁₀(RARA/MALAT1) less than 0.25, less than0.2, less than 0.15, less than 0.1, or less than 0.

In some embodiments, a subject is determined to be responsive to a RARAagonist if the strength of a RARA super enhancer in a cell in thesubject is at least 1.5-fold greater than the strength of acorresponding RARA enhancer/super enhancer in a control cell. In someaspects of these embodiments, the threshold level is at least a 2.0 foldgreater, at least a 2.5 fold greater, at least a 3 fold greater, atleast a 4 fold greater, or at least a 5 fold greater strength than inthe control cell. In some aspects of these embodiments, the strength ofa RARA super enhancer in both the test cell and the control cell arenormalized before comparison. Normalization involves adjusting thedetermined strength of a RARA super-enhancer by comparison to eitheranother enhancer or super enhancer that is native to and present atequivalent levels in both of the cells (e.g., MALAT1), or to a fixedlevel of exogenous DNA that is “spiked” into samples of each of thecells prior to super-enhancer strength determination (D A Orlando etal., Cell Rep. 2014 Nov 6, 9(3):1163-70 (2014); N Bonhoure et al.,Genome Res, 24:1157-68 (2014)).

In some embodiments, determination of whether a cell has a RARA superenhancer strength above a requisite threshold level is achieved bycomparing RARA enhancer strength in a test cell to the correspondingRARA strength in a population of cell samples, wherein each of the cellsamples is obtained from a different source (i.e., a different subject,a different cell line, a different xenograft). In some aspects of theseembodiments, only primary tumor cell samples from subjects are used todetermine the threshold level. In some aspects of these embodiments, atleast some of the samples in the population will have been tested forresponsiveness to a specific RARA agonist in order to establish: a) thelowest RARA enhancer strength of a sample in the population thatresponds to that specific RARA agonist (“lowest responder”); and,optionally, b) the highest RARA enhancer strength of a sample in thepopulation that does not respond to that specific RARA agonist (“highestnon-responder”). In these embodiments, a cutoff of RARA enhancerstrength above which a test cell would be considered responsive to thatspecific RARA agonist is set: i) equal to or up to 5% above the RARAenhancer strength in the lowest responder in the population; or ii)equal to or up to 5% above the RARA enhancer strength in the highestnon-responder in the population; or iii) a value in between the RARAenhancer strength of the lowest responder and the highest non-responderin the population.

It should be understood that in the above embodiments typically not allof the samples in a population need to be tested for responsiveness tothe RARA agonist, but all samples are measured for RARA enhancerstrength. In some embodiments, the samples are rank ordered based onRARA enhancer strength. The choice of which of the three methods setforth above to use to establish the cutoff will depend upon thedifference in RARA enhancer strength between the lowest responder andthe highest non-responder in the population and whether the goal is tominimize the number of false positives or to minimize the chance ofmissing a potentially responsive sample or subject. When the differencebetween the lowest responder and highest non-responder is large (e.g.,when there are many samples not tested for responsiveness that fallbetween the lowest responder and the highest non-responder in a rankordering of RARA enhancer strength), the cutoff is typically set equalto or up to 5% above the RARA enhancer strength in the lowest responderin the population. This cutoff maximizes the number of potentialresponders. When this difference is small (e.g., when there are few orno samples untested for responsiveness that fall between the lowestresponder and the highest non-responder in a rank ordering of RARAenhancer strength), the cutoff is typically set to a value in betweenthe RARA enhancer strength of the lowest responder and the highestnon-responder. This cutoff minimizes the number of false positives. Whenthe highest non-responder has a RARA enhancer strength that is greaterthan the lowest responder, the cutoff is typically set to a value equalto or up to 5% above the RARA enhancer strength in the highestnon-responder in the population. This method also minimizes the numberof false positives.

In some embodiments, determination of whether a cell has a RARA superenhancer above a requisite threshold level is achieved by comparing theordinal of RARA enhancer strength in a test cell to the ordinal of RARAenhancer strength in a population of cell samples, wherein each of thecell samples is obtained from a different source (i.e., a differentsubject, a different cell line, a different xenograft). In theseembodiments, at least some of the samples in the population will havebeen tested for responsiveness to a specific RARA agonist in order toestablish: a) the lowest RARA enhancer strength ordinal of a sample inthe population that responds to that specific RARA agonist (“lowestordinal responder”); and, optionally, b) the highest RARA enhancerstrength ordinal of a sample in the population that does not respond tothat specific RARA agonist (“highest ordinal non-responder”). In theseembodiments, a cutoff of RARA enhancer strength ordinal above which atest cell would be considered responsive to that specific RARA agonistis set: i) equal to or up to 5% above the RARA enhancer strength ordinalin the lowest ordinal responder in the population; or ii) equal to or upto 5% above the RARA enhancer strength ordinal in the highest ordinalnon-responder in the population; or iii) a value in between the RARAenhancer strength ordinal of the lowest ordinal responder and thehighest ordinal non-responder in the population.

It should be understood in the above embodiments, that typically not allof the samples in a population need to be tested for responsiveness tothe RARA agonist, but all samples are measured for RARA enhancerstrength and the ordinal of RARA enhancer strength compared to otherenhancers in the same sample is established. The ordinal is typicallyobtained by measuring the strength of all other enhancers in the celland determining what rank (i.e., the ordinal) in terms of strength theRARA enhancer has as compared to the other enhancers.

In some embodiments, the samples are rank ordered based on the ordinalof RARA enhancer strength. The choice of which of the three methods setforth above to use to establish the cutoff will depend upon thedifference in ordinal of RARA enhancer strength between the lowestordinal responder and the highest ordinal non-responder in thepopulation and whether the cutoff is designed to minimize falsepositives or maximize the number of responders. When this difference islarge (e.g., when there are many samples not tested for responsivenessthat fall between the lowest ordinal responder and the highest ordinalnon-responder in a rank ordering of ordinals of RARA enhancer strength),the cutoff is typically set equal to or up to 5% above the ordinal ofRARA enhancer strength in the lowest ordinal responder in thepopulation. When this difference is small (e.g., when there are few orno samples untested for responsiveness that fall between the lowestordinal responder and the highest ordinal non-responder in a rankordering of ordinal of RARA enhancer strength), the cutoff is typicallyset to a value in between the ordinal of RARA enhancer strength of thelowest ordinal responder and the highest ordinal non-responder. When thehighest ordinal non-responder has an ordinal of RARA enhancer strengththat is greater than that of the lowest responder, the cutoff istypically set to a value equal to or up to 5% above the ordinal of RARAenhancer strength in the highest ordinal non-responder in thepopulation.

In some aspects of embodiments where a test cell or sample is comparedto a population, the cutoff value(s) obtained for the population (e.g.,RARA enhancer strength or RARA enhancer ordinal) is converted to aprevalence rank and the cutoff is expressed as a percent of thepopulation having the cutoff value or higher, i.e., a prevalence cutoff.Without being bound by theory, applicants believe that the prevalencerank of a test sample will be similar regardless of the methodology usedto determine RARA enhancer strength. Thus, a prevalence cutoffdetermined for one parameter (e.g., RARA enhancer strength ordinal) isportable and can be applied to another parameter (e.g., RARA mRNA level)to determine the cutoff value for that other parameter. This allows thedetermination of a cutoff value for any parameter without having toexperimentally determine the correlation between levels of suchparameter and responsiveness to the RARA agonist. All that needs to bedetermined is what level of such other parameter corresponds to theprior determined prevalence cutoff in a population.

RARA mRNA Level Determination

Our identification of the RARA super enhancer locus allows one to useRNA transcripts to determine sensitivity instead of super-enhancer levelto determine sensitivity to a RARA agonist. RNA transcripts from thesuper-enhancer locus itself may be quantified and correlate very wellwith super-enhancer levels at that locus. We have also shown that mRNAtranscripts encoding RARA also correlate with sensitivity to RARAagonists, and thus mRNA levels can be used to identify cells that willrespond to RARA agonists.

In some embodiments, the RNA transcript level from the super-enhancerlocus is quantified using quantitative techniques that compare RARAenhancer RNA transcript levels in a sample with corresponding RARAenhancer RNA transcript levels in a cell or cell line known to benon-responsive to a RARA agonist. Such methods include RNA array orsequencing based methods for reading the eRNA associated with enhancerread through (N Hah et al., PNAS, 112(3):E297-302, 2015), as well as RNAqPCR.

In some aspects of these embodiments, at least a 1.5 fold higher RARARNA transcript level as compared to that of a corresponding RARAenhancer RNA transcript level in a cell or cell line known to benon-responsive to a RARA agonist is used as a threshold to identifysensitivity to a RARA agonist. In other aspects of these embodiments,the threshold for identifying RARA agonist responders is at least a 2.0fold higher, at least a 2.5 fold higher, at least a 3 fold higher, atleast a 4 fold higher, or at least a 5 fold higher RARA RNA transcriptlevel as compared to that in the control cell.

In some embodiments, the mRNA levels of RARA are used to identify RARAagonist responders. In one aspect of these embodiments, mRNA levels arequantified using RNA-Seq or RNA-qPCR techniques. In each of thesetechniques the RARA mRNA level is determined in a test cell and comparedto the RARA mRNA in a cell that is known to be non-responsive to a RARAagonist (the “control cell”), e.g., HCC1143 cells. In one aspect ofthese embodiments, an at least 1.5 fold higher RARA mRNA level in thetest cell as compared to the control cell indicates responsiveness toRARA agonist (i.e., the pre-determined threshold value is at least1.5-fold higher compared to the control cell). In other aspects of theseembodiments an at least 2 fold, an at least 2.5 fold, an at least 3fold, an at least 4 fold or an at least 5 fold higher RARA mRNA level inthe test cell as compared to the control cell indicates responsivenessto RARA agonist. In another aspect of these embodiments, the controlcell is any cell listed in FIGS. 3A-3M, wherein log₁₀(RARA/MALAT1) lessthan 0.25, less than 0.2, less than 0.15, less than 0.1, or less than 0.

In alternate embodiments, the RARA mRNA levels in a subject (i.e., in atumor sample, in a cancer cell sample, in a blood sample, etc.) arecompared, using the same assay, to the RARA mRNA levels in a populationof subjects having the same disease or condition to identify RARAagonist responders. In these embodiments, at least some of the samplesin the population will have been tested for responsiveness to a specificRARA agonist in order to establish: a) the lowest RARA mRNA level of asample in the population that responds to that specific RARA agonist(“lowest mRNA responder”); and, optionally, b) the highest RARA mRNAlevel of a sample in the population that does not respond to thatspecific RARA agonist (“highest mRNA non-responder”). In theseembodiments, a cutoff of RARA mRNA level above which a test cell wouldbe considered responsive to that specific RARA agonist is set: i) equalto or up to 5% above the RARA mRNA level in the lowest mRNA responder inthe population; or ii) equal to or up to 5% above the RARA mRNA level inthe highest mRNA non-responder in the population; or iii) a value inbetween the RARA mRNA level of the lowest mRNA responder and the highestmRNA non-responder in the population.

In some embodiments not all of the samples in a population need to betested for responsiveness to the RARA agonist, but all samples aremeasured for RARA mRNA levels. In some embodiments, the samples are rankordered based on RARA mRNA levels. The choice of which of the threemethods set forth above to use to establish the cutoff will depend uponthe difference in RARA mRNA levels between the lowest mRNA responder andthe highest mRNA non-responder in the population and whether the cutoffis designed to minimize false positives or maximize the potential numberof responders. When this difference is large (e.g., when there are manysamples not tested for responsiveness that fall between the lowest mRNAresponder and the highest mRNA non-responder in a rank ordering of RARAmRNA levels), the cutoff is typically set equal to or up to 5% above theRARA mRNA level in the lowest mRNA responder in the population. Whenthis difference is small (e.g., when there are few or no samplesuntested for responsiveness that fall between the lowest mRNA responderand the highest mRNA non-responder in a rank ordering of RARA mRNAlevels), the cutoff is typically set to a value in between the RARA mRNAlevels of the lowest mRNA responder and the highest mRNA non-responder.When the highest mRNA non-responder has a RARA mRNA levels that isgreater than the lowest mRNA responder, the cutoff is typically set to avalue equal to or up to 5% above the RARA mRNA levels in the highestmRNA non-responder in the population.

In some embodiments the population is rank ordered based on RARA mRNAlevel. In these embodiments, the RARA mRNA level in each sample ismeasured and compared to the mRNA levels of all other mRNAs in the cellto obtain an ordinal ranking of the RARA mRNA level. A cutoff based onRARA mRNA ordinal ranking is then determined based on samples in thepopulation tested for responsiveness to a RARA agonist in the samemanner as described previously for determining a RARA super enhancerstrength ordinal cutoff. The determined RARA mRNA ordinal cutoff is thenused either directly or to determine a prevalence cutoff, either ofwhich is then used to stratify additional samples for potentialresponsiveness to the RARA agonist.

In some embodiments, the cutoff for RARA mRNA levels is determined usingthe prevalence cutoff established based on RARA enhancer strength orRARA enhancer strength ordinal, as described above. In some aspects ofthese embodiments, a population is measured for mRNA levels and theprior determined prevalence cutoff is applied to that population todetermine a mRNA cutoff level. In some aspects of these embodiments arank-order standard curve of RARA mRNA levels in a population iscreated, and the pre-determined prevalence cutoff is applied to thatstandard curve to determine the RARA mRNA cutoff level.

In some aspects of embodiments where a test cell or sample is comparedto a population, the cutoff mRNA level value(s) obtained for thepopulation is converted to a prevalence rank and the mRNA level cutoffis expressed as a percent of the population having the cutoff value orhigher, i.e., a prevalence cutoff

Without being bound by theory, applicants believe that the prevalencerank of a test sample and the prevalence cutoff in a population will besimilar regardless of the methodology used to determine RARA mRNAlevels.

In some aspects of these embodiments, a subject is identified as a RARAagonist responder if its RARA mRNA level corresponds to a prevalencerank in a population of 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%,70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%,56%, 55%, 54%, 43%, 42%, 51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%,42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%,28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, or 20% as determined by RARAmRNA levels in the population. In one aspect of these embodiments, thecutoff value is established based on the prevalence cutoff establishedfor RARA enhancer strength. In an alternate aspect of these embodiments,the cutoff value is established based on the prevalence cutoffestablished for RARA enhancer strength ordinal. In another alternateaspect of these embodiments, the cutoff value is established based onRARA mRNA levels. In more specific aspects of these embodiments, acutoff value for breast cancer patients is established based on theprevalence cutoff determined for RARA enhancer strength ordinal, andthat prevalence cutoff value is used as to determine the cutoff valuefor RARA mRNA levels In even more specific aspects of these embodiments,the cutoff value for breast cancer patients is the value determinedusing a prevalence value of between 50% and 60%, e.g., 50-55%, 55-60%,50-56%, 50-57%, 51-55%, 51-56%, 51-57%, 52-55%, 52-56%, 52-57%, 53-55%,54-56%, 53-56%, or 54-55%. In still other more specific aspects of theseembodiments, the cutoff value is set using a prevalence value of 55% orof 56%. In other more specific aspects of these embodiments, a cutoffvalue for AML patients is established based on the prevalence valuedetermined for RARA enhancer strength ordinal, and that prevalence valueis used to determine the cutoff value for RARA mRNA levels In even morespecific aspects of these embodiments, the cutoff value for AML patientsis determined using a prevalence cutoff of between 25-45%,e.g., between25-30%, 25-35%, 25-40%, 30-35%, 30-40%, 35-45%, 35-40%, 31-35%, 32-35%,33-35%, 34-35%, 31-36%, 32-36%, 33-36%, 34-36%, or 35-36%. In other evenmore specific aspects of these embodiments, the cutoff value for AMLpatients is determined using a prevalence value of 36%. In yet othereven more specific aspects of these embodiments, the cutoff value forAML patients is determined using a prevalence value of 25%.

In still other embodiments, a population may be divided into threegroups—responders, partial responders and non-responders—and two cutoffvalues or prevalence cutoffs are set. The partial responder group mayinclude responders and non-responders, as well as those populationmembers whose response to a RARA agonist was not as high as theresponder group. In these embodiments, two cutoff values or prevalencecutoffs are determined. This type of stratification may be particularlyuseful when in a population the highest RARA mRNA non-responder has aRARA mRNA level that is greater than the lowest RARA mRNA responder. Inthis scenario the cutoff level or prevalence cutoff between respondersand partial responders is set equal to or up to 5% above the RARA mRNAlevel of the highest RARA mRNA non-responder; and the cutoff level orprevalence cutoff between partial responders and non-responders is setequal to or up to 5% below the RARA mRNA level of the lowest RARA mRNAresponder. The determination of whether partial responders should beadministered the RARA agonist will depend upon the judgment of thetreating physician and/or approval by a regulatory agency.

Methods of quantifying specific RNA sequences in a cell or biologicalsample are known in the art and include, but are not limited to,fluorescent hybridization such as utilized in services and productsprovided by NanoString Technologies, array based technology(Affymetrix), reverse transcriptase qPCR as with SYBR® Green (LifeTechnologies) or TaqMan® technology (Life Technologies), RNA sequencing(e.g., RNA-seq), RNA hybridization and signal amplification as utilizedwith RNAscope® (Advanced Cell Diagnostics), or northern blot.

In some aspects of these embodiments, the level of RNA transcript(either mRNA or another RARA transcript) in both the test cell and thecontrol cell or all members of the population are normalized beforecomparison. Normalization involves adjusting the determined level of aRARA RNA transcript by comparison to either another RNA transcript thatis native to and present at equivalent levels in both of the cells(e.g., GADPH mRNA, 18S RNA), or to a fixed level of exogenous RNA thatis “spiked” into samples of each of the cells prior to super-enhancerstrength determination (J Lovén et al., Cell, 151(3):476-82 (2012); JKanno et al., BMC Genomics 7:64 (2006); J Van de Peppel et al., EMBO Rep4:387-93 (2003)).

Cancers and Other Diseases

The methods and packaged pharmaceuticals of the present invention aretheoretically useful to treat any cancer that is characterized by theassociation of a super enhancer with a RARA gene in such cancer. Superenhancer-associated RARA genes may be more prevalent in certain types ofcancers than others. The inventors have discovered RARA associated superenhancers in cancers of the skin, breast, blood, bone, cervix, colon,rectum, esophagus, lymph node, lung, ovary, uterus, pancreas, prostate,kidney, and spleen; and in particular in AML (especially in non-APL AMLand in other forms of AML that are not characterized by a chromosomaltranslocation involving a RARA gene), myelodysplastic syndrome (MDS),colorectal, HER2+ breast, ER+ breast, triple-negative breast cancer,glioblastoma, glioma, gastric cancer, renal clear cell carcinoma,non-small cell lung cancer, melanoma, multiple myeloma, pancreaticcarcinoma, pheochromocytoma, paraganglioma, and prostate adenocarcinoma.Without being bound by theory, the inventors believe that SE-associatedRARA genes will be found in subsets of all cancers and that subjectswithin those subsets will be more responsive to a RARA agonist thanother subjects having the same type of cancer without a SE-associatedRARA gene.

We also believe that the discovery of SE-associated RARA in tissues innon-cancer disease suggests potential for the effective use of RARAagonists to treat such diseases. We have detected the presence of theRARA super enhancer in adipose tissue suggesting a method of identifyingpatients suffering from diabetes or obesity who may be effectivelytreated with a RARA agonist. Retinoic acid has an established role inthe differentiation of adipose tissue (J Mercader, et al.,Endocrinology, 147(100):5325-5332, 2006). In addition, a relationshipbetween PPAR-γ and RARA has been shown (A Redonnet, et al., Int JObesity, (26)920-927, 2002).

A super enhancer associated with the RARA locus has also been detectedin numerous types of hematological cells. This includes CD133⁺hematopoietic stem cells, CD14⁺ monocytes, CD19⁺ early B-cells, CD20B-cells, CD3⁺ mature T-cells, CD34⁺ hematopoietic progenitors, CD4⁺ Thelper cells, CD56 Natural Killer cells, and CD8⁺ cytotoxic T cells. Thepresence of RARA associated super-enhancers in these cells suggest thatRARA agonists may be useful to treat subsets of patients suffering fromcertain autoimmune diseases, including but not limited to psoriasis,multiple sclerosis, rheumatoid arthritis, ankylosing spondylitis, celiacdisease, myasthenia gravis, systemic lupus erythematosus andscleroderma.

Cell from patients suffering from Autosomal Dominant Polycystic KidneyDisease also has been found to have a RARA associated super enhancer andthus could be candidates for effective treatment with RARA agonist.There is some evidence that ADPKD can respond to retinoids (Q Qian, etal., Kidney International, (59): 2005-2022, 2001), so identifying thosepatients who also have a SE associated with the RARA locus may be usedas a stratification method to better select those patients who are morelikely to respond to a RARA selective agonist.

In some embodiments, the disease to be treated in the methods and by thepackaged pharmaceutical compositions of the invention is cancer. In someaspects of these embodiments, the disease to be treated is selected frombreast cancer, glioblastoma, neuroblastoma and AML. In some morespecific aspects of these embodiments, the disease to be treated isselected from breast cancer, glioma, cervical and endocervicalcarcinoma, colon and rectal adenocarcinoma, head and neck squamouscarcinoma, kidney renal papillary cell carcinoma, lung adenocarcinoma,pancreatic adenocarcinoma, pheochromocytoma, paraganglioma, skincutaneous melanoma, uterine carcinoma, MDS and AML. In some morespecific aspects of these embodiments, the disease to be treated isselected from breast cancer, MDS and AML. In some more specific aspectsof these embodiments, the disease to be treated is selected from breastcancer and AML. In even more specific aspects of these embodiments, thedisease to be treated is non-APL AML or an AML that is not characterizedby a chromosomal translocation involving a RARA gene.

In some embodiments, the subject to be treated with a RARA agonist(e.g., tamibarotene) is suffering from relapsed or refractory AML. Asubject is classified as having relapsed or refractory AML if they: a)do not demonstrate a partial response after a first cycle of inductionchemotherapy; or b) do not demonstrate a complete response after asecond cycle of induction chemotherapy; or c) relapse after conventionalchemotherapy; or d) relapse are undergoing a single stem celltransplantation.

In some embodiments, the subject to be treated with a RARA agonist(e.g., tamibarotene) is suffering from refractory MDS. A subject isclassified as having refractory MDS if they: a) are categorized ashaving high risk or intermediate-2 MDS (as determined using theInternational Prognostic Staging System (“IPPS”)) and have failed toachieve any hematologic improvement (as measured by IWG 2006 criteria)after at least 4 cycles of induction therapy with hypomethylating agents(e.g., azacitidine, decitabine), or has relapsed after any duration ofcomplete or partial response; orb) are categorized as IPSSintermediate-1 or low-risk MDS and are either transfusion dependent orhave failed treatment with erythropoiesis stimulating agents (ESA).

In other embodiments, the subject to be treated with a RARA agonist(e.g., tamibarotene) is an elderly unfit subject. The term “elderlyunfit” as used herein means the subject is a human at least 60 years ofage and who is determined by a physician to not be a candidate forstandard induction therapy.

RARA Agonists

The choice of RARA agonist with which to treat a patient identified ashaving a super enhancer associated with a RARA gene may be made from anyRARA agonist known in the art. It is preferable that the RARA agonistutilized in the methods of the invention be specific for RARA and havesignificantly less (at least 10× less, at least 100× less, at least1,000× less, at least 10,000× less, at least 100,000× less) agonisticactivity against other forms of RaR, e.g., RaR-ß and RaR-γ.

In some embodiments, the RARA agonist is selected from a compounddisclosed in or any compound falling within the genera set forth in anyone of the following United States patents: U.S. Pat. Nos. 4,703,110,5,081,271, 5,089,509, 5,455,265, 5,759,785, 5,856,490, 5,965,606,6,063,797, 6,071,924, 6,075,032, 6,187,950, 6,355,669, 6,358,995, and6,387,950, each of which is incorporated by reference.

In some embodiments, the RARA agonist is selected from any of thefollowing known RARA agonists set forth in Table 1, or apharmaceutically acceptable salt thereof, or a solvate or hydrate of theforegoing:

TABLE 1 Exemplary RARA Agonists useful in the invention. Structure CodeName(s)

Am-580; CD- 336; Ro-40- 6055

AM-80; INNO-507; NSC-608000; OMS-0728; TM-411; TOS- 80; TOS-80T; Z-208;tamibarotene

Am-555S; TAC-101; amsilarotene

ER-34617

ER-38930

ER-65250

ER-38925

ER-35368

E-6060

ER-41666

AGN-195183; NRX-195183; VTP-195183; VTP-5183 IRX-5183

BMS-228987

BMS-276393

BMS-231974

ABPN; CBG-41

PTB

A-112

BD-4; BJ-1

Tazarotene; AGN-190168

Ch-55In some embodiments, the RARA agonist is tamibarotene. In someembodiments, the RARA agonist is

Packaged Pharmaceutical Compositions

The packaged pharmaceutical compositions of the present inventioncomprise a written insert or label comprising instructions to use theRARA agonist in a subject suffering from a cancer and who has beendetermined to have a super enhancer associated with a RARA gene having astrength, or ordinal rank equal to or above a threshold level, or a RARAmRNA level equal to or above a threshold level. As described in detailabove, the threshold level is determined in a population of samples fromeither subjects diagnosed as suffering from the same disease or celllines or xenograft models of the same disease as that for which thepharmaceutical composition is indicated for treatment. The instructionsmay be adhered or otherwise attached to a vessel comprising the RARAagonist. Alternatively, the instructions and the vessel comprising theRARA agonist will be separate from one another, but present together ina single package, box or other type of container.

The instructions in the packaged pharmaceutical composition willtypically be mandated or recommended by a governmental agency approvingthe therapeutic use of the RARA agonist. The instructions may comprisespecific methods of determining whether a super enhancer is associatedwith a RARA gene, as well as quantification methods to determine whetheran enhancer associated with a RARA gene is a super enhancer,quantification methods to determine RARA mRNA levels; and/or thresholdlevels of super enhancers or RARA mRNA at which treatment with thepackaged RARA agonist is recommended and/or assumed therapeuticallyeffective. In some aspects, the instructions direct that the compositionbe administered to a subject whose RARA mRNA level falls in at least the30^(th) percentile of a population whose RARA mRNA levels have beenmeasured. In some aspects of these embodiments, a subject is identifiedas a RARA agonist responder if its RARA mRNA level prevalence rank is79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%,65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 43%, 42%,51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%,37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%,23%, 22%, 21%, or 20% in a population whose RARA mRNA levels have beenmeasured. In some aspects, the instructions direct that the compositionbe administered to a subject whose RARA mRNA level as measured by aspecific assay

The instructions may optionally comprise dosing information, the typesof cancer for which treatment with the RARA agonist was approved,physicochemical information about the RARA agonist; pharmacokineticinformation about the RARA agonist, drug-drug interaction information.In some aspects, the instructions direct that the composition beadministered to a subject diagnosed as suffering from non-APL AML. Inother aspects, the instructions direct that the composition beadministered to a subject diagnosed as suffering from breast cancer. Insome aspects, the instructions direct that the composition beadministered to a subject diagnosed as suffering from MDS. In someaspects, the pharmaceutical composition comprises tamibarotene. In someaspects, the pharmaceutical composition comprises AGN-195183.

EXAMPLES

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. The synthetic andbiological examples described in this application are offered toillustrate the compounds, pharmaceutical compositions, and methodsprovided herein and are not to be construed in any way as limiting theirscope.

Example 1. ChIP-seq Analysis to Identify RARA Associated Super Enhancer

I. Cross-linking of cells. For cultured cells, we typically cross-linkbetween 5×10⁷ and 1×10⁸ cells at a time (equivalent to 70-80% confluencyfor adherent cells in 8-12 15 cm² plates or suspension cells in 8-12 175cm² flasks). We use 20-50 million cells for each location analysisreaction. For adherent cells, we add 1/10 volume of fresh 11%formaldehyde solution (0.1M NaCl, 1 mM EDTA, pH 8, 0.5 mM EGTA pH 8, 50mM Hepes) to plates, swirl the plates briefly and let them sit at roomtemperature (“RT”) for 8 min. We then add 1/20 volume of 2.5M glycine or½volume of 1M Tris pH 7.5 to quench formaldehyde and incubate for atleast 1 min. We then rinse the cells 3× with 20 ml cold 1×phosphate-buffered saline (“PBS”); harvest the cells using siliconscraper; and spin the cells at 2k for 10 minutes at 4° C. in tabletopcentrifuge. Cells are then transferred to 15 ml conical tubes and spunat 2 k for 10 minutes at 4° C. The pelleted cells are flash frozen inliquid nitrogen and stored at −80° C.

For cells in suspension we add 1/10 volume of fresh 11% formaldehydesolution to cell suspension, mix and let the mixture sit at RT for 8min. We then add 1/20 volume of 2.5M glycine or ½ volume of 1M Tris pH7.5 to quench formaldehyde and incubate for at least 1 min. We thenrinse the cells 3× with 20-50 ml cold 1× PBS, centrifuging for 5min at2,000 RPM to pellet the cells before and after each wash. Cells are thentransferred to 15 ml conical tubes and spun at 2k for 5 minutes at 4° C.The supernatant is removed, residual liquid is removed by dabbing withKIMWIPES® (absorbent tissues) and then the pelleted cells are flashfrozen in liquid nitrogen and stored at −80° C.

For cells derived from primary blood, we cross-link between 2×10⁵ and1×10⁷ cells per sample by addition of 1/10 volume of fresh 11%formaldehyde solution (0.1M NaCl, 1 mM EDTA, pH 8, 0.5 mM EGTA pH 8, 50mM Hepes), cross linking is allowed to proceed at RT for 8 min. We thenadd 1/20 volume of 2.5M glycine or ½ volume of 1M Tris pH 7.5 to quenchformaldehyde and incubate for at least 1 min. We then rinse the cells 3×with 1-2 ml cold 1× PBS and harvest the cells by centrifugation. Cellpellets are then directly subjected to ChIP-seq analysis as described.

For cells derived from primary solid tissue, we use 250-500 ug of frozentissue per ChIP. Frozen tissues are diced with razor blade (on coldsurface, <−80 C) and scissors for 2 min in 1% Formaldehyde solution (1%Formaldehyde; 0.1M NaCl, 1 mM EDTA, pH 8, 0.5 mM EGTA pH 8, 50 mMHepes). Tissues are chopped to a fine slurry for 2 min and to the slurryis added 9 ml of 1% formaldehyde solution. Crosslinking is allowed toproceed to a total time of 8 min. We then add 1/20 volume of 2.5Mglycine or 1/2 volume of 1M Tris pH 7.5 to quench formaldehyde andincubate for at least 1 min. We then rinse the cells 3× with 1-2 ml cold1× PBS and harvest the cells by centrifugation. Cells are resuspended in6 ml PBS (Containing Complete™ protease inhibitors) and douncehomogenized ˜40× with loose pestle homogenizer. Cells are recovered bycentrifugation at 3,000 RPM for 2 min. Supernatant is removed andpelleted cells flash frozen in liquid nitrogen for ChIP-seq analysis asdescribed.

II. Binding of Antibody to magnetic beads. We use 60 μL of Dynabeads®Protein G per 2 ml immunoprecipitate (Invitrogen). Beads are washed 3times for 5 minutes each with 1.0 ml blocking buffer (0.5% BSA w/v inPBS) in a 1.5 ml Eppendorf tube. A magnet (Invitrogen) is used tocollect the beads (we allow magnet binding for at least 1 full minute)after each wash and the supernatant is then aspirated. The washed beadsare resuspended in 250 ul blocking buffer to which 6μg of antibody isadded and the mixture is allowed to incubate with end-over-end mixingovernight (minimum 6 hours). The antibody-bound-beads are washed 3× for5 min each with 1 ml blocking buffer and resuspended in blocking buffer(60 ul per IP). These last washes and resuspensions are done once thecells have been sonicated (see next step) and just prior to overnightimmunoprecipitation.

III. Cell Prep and Genomic Fragmentation. We add 1× protease inhibitors(Complete, Roche; prepare by dissolving one tablet in 1 ml H₂O for 50×solution and store in aliquots at −20° C.) to all lysis buffers beforeuse. Each tube of cells (approximately 5×10⁷ cells) is resuspended in5-10 ml of lysis buffer 1 (LB1; 140 mM NaCl, 1 mM EDTA, 10% glycerol,0.5% NP-40, 0.25% Triton X-100) and rocked at 4° C. for 10 minutes. Thecells are centrifuged at 2,000 RPM×5 min in tabletop centrifuge at 4° C.and the supernatant aspirated off. The cells are resuspended in 5 mlLysis Buffer 2 (LB2; 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH8) and incubated end over end at 4° C. for 10 minutes. The cells areagain pelleted at 2,000 RPM×5 min in tabletop centrifuge at 4° C. andwashed in 2-5 ml Covaris sonication buffer (10 mM Tris pH 8.0, 1 mMEDTA, 0.1% SDS). Pellets are spun down at 2,000 RPM×5min in tabletopcentrifuge at 4° C. The cells are pelleted at 2,000 RPM×5 min intabletop centrifuge at 4° C. and resuspended at a concentration of 20-50million cells/1 ml of Covaris sonication buffer.

One ml of cell lysate is put into 12×12 Covaris micro tubes andsonicated for 5 minutes total (Peak power 140, duty factor 5.0,cycles/burst 200). We recombine sonicates into one 15 ml tube, add 1volume of 2× Dilution mix (300 mM NaCl, 2 mM EDTA, 50 mM Tris pH 8.0,1.5% Triton-X, 0.1% SDS), pellet the insoluble fraction at 14,000 RPMfor 10 min at 4° C. and collect the supernatant into a single tube. Thesupernatant is used as ChIP input for chromatin immunoprecipitations. Wealso collect 50 μL of the cell lysate for whole cell extract (“INPUT”)sample control.

IV. Chromatin Immunoprecipitation. Fifty μL of antibody-conjugated beadsfrom step II is added to cleared cellular extract (Prepared in Step III)solution in 1.5 ml tubes and rocked overnight at 4° C. (minimum 8 hours)to immunoprecipitate DNA-protein complexes.

V. Wash, elution, and cross-link reversal. All buffers used in thesesteps are ice cold. We use a magnetic stand to precipitate magneticbeads, washed 3 times 5 minutes each with gentle end over end mixingwith 1 ml Wash Buffer 1 (50 mM HEPES pH 7.5; 140 mM NaCl; 1 mM EDTA; 1mM EGTA; 0.75% Triton-X; 0.1% SDS; 0.05% DOC); wash once for 5 minuteswith 1 ml Wash Buffer 2 (50 mM HEPES pH 7.5; 500 mM NaCl; 1 mM EDTA; 1mM EGTA; 0.75% Triton-X; 0.1% SDS; 0.05% DOC); and once for 5 minuteswith 1 ml Wash Buffer 3 (10 mM Tris pH 8.0; 1 mM EDTA; 50 mM NaCl). Weaspirate all residual wash buffer, spin the beads gently at 2,000 RPMfor 1 min; put tubes back onto the magnet and remove all traces ofbuffer. We then add 210 μl of Elution buffer (50 mM Tris pH8; 10 mMEDTA; 1% SDS) and elute at 65° C. for 60 min with brief vortexing toresuspend beads every 15 min. We separate the beads from the supernatantusing the magnet; remove 200 μL of supernatant and place in a clean tubefor reverse cross-linking. We reverse x-link both IP and whole cellextract fractions overnight at 65° C. (minimum 8 hours, but maximum 18hours). We then use heating to separately reverse cross-linked both thesample for immunoprecipitation and the whole cell extract fractions byincubating overnight at 65° C. (minimum 8 hours, but maximum 18 hours).The heating facilitates the hydrolysis of the formaldehyde cross-links.

VI. Cleanup and purification of DNA. We add 2000 of TE (50 mM Tris pH8;1 mM EDTA) and 2.7 μl of 30 mg/ml RNaseA (0.2 mg/ml final concentration)to each sample, mixed and incubate at 37° C. for 2 hours. We then add 5μl of calcium chloride solution (300 mM CaCl₂ in 10 mM Tris pH8.0) toeach sample along with 44, of 20 mg/ml proteinase K (0.2 mg/ml finalconcentration), mix and incubate at 55° C. for 60 minutes. We then add4000 of phenol:chloroform:isoamyl alcohol at 25:24:1 ratio (SigmaAldrich #P3803) to each tube, mix on a vortex mixer on low setting(5/10) and invert each tube to mix further.

We prepare a PhaseLock Gel™ tube (Qiagen, 3 Prime) for each sample byspinning the tube at room temperature for 30 seconds at 10,000 RPM. Wenext add the sample DNA sample in phenol:chloroform:isoamyl alcohol tothe PhaseLock Gel tube and spin at 12,000-16,000×g for 2 minutes at roomtemperature. We transfer the aqueous solution to a new 1.6 ml tube (topfraction), add 20 ul of 5M NaCl, and 1.5 ul of 20 ug/ul glycogen (30 ugtotal), then add 1 ml of EtOH and mix by vortex or inversions. Thesample is then incubated at −20° C. overnight (6-16 hours). We then spinthe mixture at 20,000×g for 20 minutes at 4° C. to pellet the DNA,remove the supernatant with 1 ml pipette tip, wash the pellets in 8000of 80% EtOH, spin at 20,000×g for 20 minutes at 4° C. and remove thesupernatant with 1 ml pipette tip. We again spin the sample for 1 min at20,000×g, remove all traces of the supernatant and let air dry for 5-20minutes. The pellets should not have a halo of water around them andshould be glassy or flaky dry. We then dissolve the pellet in 60 μl ofwater, using 10 μl for qPCR validation and 500 for sequencing. ForChIP-qPCR, amplification is performed using 1 uL of the sample per wellwith 0.8 uM of forward (F) and reverse (R) primers each mixed with 2×Power SYBR Green PCR Master Mix from Life Technologies following themanufacturer's protocol. Primers to use are as shown in the table below,where V1, V2, and V3 are primer pairs designed to hybridize to differentregions within the RARA enhancer and “down” is a primer pair designed tohybridize to downstream non-acetylated, non-gene coding control region.

V1 V3 F AAACGTGTCCCCACCTCTC F TTCCTAGTGGTCCCCCTTCC (SEQ ID NO: 1)(SEQ ID NO: 5) R CCAGCCAGGCACATAGGG R TGAAGATTGTTTGCACCCCCT(SEQ ID NO: 2) (SEQ ID NO: 6) V2 down F GTCACCGCACTCACTTCCAT FCTGCTGGTACCCAGAAGTGAG (SEQ ID NO: 3) (SEQ ID NO: 7) RAAATAGCGCTCGGTGGAGAA R TGTTGAGTTTTGCCAGTCTCTT (SEQ ID NO: 4)(SEQ ID NO: 8)

The results of ChIP-qPCR for a strongly responsive (Au565) and a weaklyresponsive (T47D) cell line as compared to a whole cell extract (“WCE”)from each cell is shown in FIGS. 1A-B. This figure illustrates thatthese primers are effective at measuring a strong enrichment for theH3K27ac mark at the enhancer for these cell lines in the pull down butnot WCE after normalization to a nearby intergenic control region.

VII. ChIP Seg Analysis and Quantification of RARA super enhancer. We usethe above-described methodology using H3K27ac ChIP-seq to identify asuper enhancer locus overlapping the RARA gene atchr17:38458152-38516681 (genome build hg19). This chromosome region wasdesignated by consensus over the samples that are examined, but couldvary somewhat based on which genomic marker is being detected or thetype of tissue being used. To assess the strength of this super-enhancerlocus across different samples we perform H3K27ac ChIP-seq in eachsample and also sequence a match ChIP Input sample. All H3K27ac andinput samples are aligned to the hg19 genome using Bowtie2 (using thesensitive parameter). The gene track visualization shown in FIG. 2demonstrates a representative sample of cells showing the counts ofaligned reads in 1 bp bins, where each read is extended 200bp in thedirection of the alignment. Read counts are in number of alignedreads-per-million aligned reads (RPM). AUC is calculated by summing thereads at each base pair within a defined locus. Based on the location ofthe RARA super enhancer locus, we also assessed the level of RARA superenhancer relative to the MALAT1 super enhancer locus in existing H3K27acChIP-seq maps from various cell lines and patient samples.

For each H3K27Ac/Input sample pair (rows in the table in FIGS. 3A-3M),the number of H3K27Ac or Input reads aligning to either the RARAsuper-enhancer (chr17:38458152-38516681) or the positive controlsuper-enhancer at MALAT1 (chr11:65263724-65266724) are counted. All readcounts are in RPM. We then subtract the Input signal from the H3K27Acsignal at both loci to get the enrichment of H3K27ac ChIP-seq reads overbackground (DIFF column). Finally, to assess the strength of the RARAsuper-enhancer relative to the MALAT1 positive control super-enhancer,we calculate the ratio of the RARA super-enhancer H3K27Ac enrichmentsignal to the H3K27Ac enrichment signal at the MALAT1 super-enhancer andreport this ratio in the RARA/MALAT1 column. A higher value indicated astronger RARA super-enhancer score. We also calculate thelog₁₀(RARA/MALAT1) ratio. All of these values for each sample tested areshown in FIGS. 3A-3M.

The log₁₀ ratio of RARA/MALAT1 for all of the breast cancer and AML celllines and patient samples are graphed as shown in FIGS. 4A and 4B,respectively. The 0.4 log₁₀ threshold shown in each graph is the lowestSE strength found to be sensitive to tamibarotene in in vitro celllines. As can be seen in these graphs, a certain percentage of samplesof each cancer type falls above this threshold. FIG. 5 shows the log₁₀ratio of RARA/MALAT1 for normal hematological cell lines and patientsamples tested. As can be seen, a certain percentage of samples of eachhematological cell tested falls above the threshold, suggesting thatthis type of patient stratification would be useful to identify subjectswith hematological disorders that would be responsive to a RARA agonist.

RARA SE rank is calculated using ROSE as described in U.S. Pat. No.9,181,580, the disclosure of which is herein incorporated by reference.First, ROSE is used to calculate the enhancer scores for all of thebreast cancer and AML cell lines and patient samples. Within eachsample, the enhancers are ranked by score, and the rank of the RARAsuper-enhancer is the rank of the enhancer overlapping the RARA gene(chr17:38465444-38513094) with the highest score.

FIG. 6 shows a comparison of the log₁₀ ratio of RARA/MALAT1 for celllines versus patient samples in breast cancer and AML. As can be seen inthis figure, patient samples in both cancer types have a statisticallysignificant higher level RARA than corresponding cell lines. Thisdemonstrates that high RARA super enhancer levels are not an in vitrophenomenon limited to cell lines. In addition, this illustrates that ahigher proportion of the patient samples would be expected to show aresponse to RARA agonist than cell lines.

Example 2. Screening of Various RaR Modulator Compounds against BreastCell Cancer Panel

I. Materials. All cell lines are obtained from ATCC and cultured at 37°C. in 5% CO₂. All are grown in RPMI1640 supplemented with 10 mM HEPESbuffer, 2 mM L-glutamine, 50 U/mL penicillin, 50U/mL streptomycin and10% fetal bovine serum (FBS, all from Invitrogen). Cell lines includedAU565, SKBR3, T47D, HCC1143, MCF7, ZR-75-1, and HCC1569.

Tamibarotene, AM580, and tretinoin are obtained from Sigma Aldrich.Tazarotenic acid is obtained from Carbosynth. Adapalene, BMS195614,BMS493, and BMS961 are obtained from Tocris. Etretinate is obtained fromSanta Cruz. Tazarotene is obtained from Selleckchem.

The agonist specificity of the various test compounds for each type ofRaR is assessed at Life Technologies using their SelectScreen® NuclearProfiling Services. Obtained values are shown in the table below:

RAR-a/RAR-g Stated RAR alpha RAR beta RAR gamma Selectivity Compoundspecificty EC50 % max EC50 % max EC50 % max EC50 % max Adapalene RAR5.011 102.2 9.732 69.36 17.26 87.7 3.4444 1.1653 BMS753 RARa 1.434 122.2700.8 6 136 19.83 94.8396 6.1624 Tamibarotene RARa 0.2334 187.6 417.8114.9 48.59 106.1 208.1834 1.7681 AM580 RARa 0.2515 199 144.8 78.244.951 87.06 19.6859 2.2858 Tazarotenic RAR 0.3532 121.4 5.171 71.881.101 78.33 3.1172 1.5499 Tretinoin RA 1.095 149.4 1.076 92.77 1.73299.37 1.5817 1.5035 BMS961 RARg 1169 53.22 33510 149.8 3.74 114.2 0.00320.4660 BMS493 Antagonist 27.42 −3.417 0 0 511.5 −13.41 BMS195614Antagonist 1339 −31.01 0 0 433.6 −14.57As can be seen in the above table tamibarotene, BMS753 and AM580 havethe greatest specificity for RARA over RaR-γ, thus confirming theirstatus as RARA specific agonists. This specificity is important becauseagonism of RaR-γ is associated with toxicity. Agonism of RaR-B is notknown to contribute to efficacy or toxicity and therefore should notaffect the therapeutic potential of an agonist.

II. Medium Throughput Screening. On the day of the experiment, cells arehomogenized using Accumax (EMD Millipore), counted, and adjusted to40,000 cells/ml for breast cancer lines and 60,000 cells/mL for AML inappropriate growth media. Using a Biotek EL406, 50 μl of cells aredistributed into white (ATPlite) or black (CyQuant) 384-well plates(Thermo). Cells are returned to 37° C. incubator to allow adhesion.After three hours, compounds are added to plates using a 20 nl 1384-wellpin transfer manifold on a Janus workstation. Stocks are arrayed in 10point quadruplicate dose response in DMSO stock in 384-well compoundplates. After addition of compound, plates are incubated for five or tendays in a 37° C. incubator.

Cell viability is read out using ATPlite (Perkin Elmer) or CyQuant (LifeTechnologies). For ATPlite, plates are removed from the incubator andbrought to room temperature prior to use. Lyophilized powder of ATPlitereagent is resuspended in lysis buffer and diluted 1:2 with distilledwater. 25 μL of this solution is added to each well using the Biotekliquid handler. Plates are incubated for 15 min at room temperaturebefore the luminescence signal is read on an Envision Plate Reader(Perkin Elmer). For CyQuant, reagents are mixed as per manufacturer'sinstructions in PBS (Gibco). Reagent are added using a multichannelpipet and plates are replaced in incubator for 30 minutes prior toreadout on an Envision Plate Reader (Perkin Elmer).

Data acquired as described is stored and grouped in Microsoft's Exceland analyzed using GraphPad Prism Software. Curve fits to calculate EC50and Emax are done in GraphPad Prism version 6.0 using four parameter(Hill slope not assumed to be equal to 1) non-linear regressions withthe log10 transformed data of the compound concentrations plottedagainst the percent viability of the cells when normalized to DMSO onlytreated wells included on the plate. Edge wells were excluded.

As shown in FIG. 7 , four different breast cell cancer lines exhibitdifferent responses to tamibarotene, with a sensitivity order ofSKBR3>Au565>ZR75>Hcc1143. This correlates very well with the level ofsuper-enhancer at the RARA site as shown in FIG. 2 . The correlation ofsensitivity to tamibarotene to super-enhancer strength (log₁₀ EC₅₀ vsRARA/MALAT1 super enhancer ratio) for 7 different breast cell cancerlines is shown in FIG. 8 to have a correlation (R²) value of 0.7086.

As shown in FIG. 9 , three different AML cell lines exhibit differentresponses to tamibarotene, with a sensitivity order of SigM5>OCI M1>HEL.As shown in FIGS. 23A-B, two additional AML cell lines, MV411 andKasumi, are assayed along with SigM5 and HEL for sensitivity totamibarotene and demonstrated a sensitivity order ofSigM5>MV411>HEL=Kasumi. The correlation of sensitivity to tamibaroteneto super enhancer strength (log₁₀ EC₅₀ vs RARA/MALAT1 super enhancerratio (“IEA”) for 11 different AML cell lines is shown in FIG. 10 tohave a correlation (R²) value of 0.3635, but that value increases to0.5680 when one cell line that shows significant deviation from the norm(higher SE but lower than expected sensitivity), HEL, is not taken intoaccount.

Example 3. Measurement of RARA RNA and Protein Expression Levels

Expression level measurements are employed to ascertain the level ofmRNA for the RARA gene tagged. mRNA levels correlate well with enhancerlevels and therefore are also predictive sensitivity to RARA agonists.We use various means of measuring RNA as set forth below.

I. Array-Based Technology. Expression levels in HCC1143 and AU565 areassessed with triplicate batches of 1×10⁶ cells. RNA is extracted fromcells using Trizol® and purified using the mirVana™ RNA purification kit(both from Life Technologies), following the manufacturer's protocol.RNA levels are read out on Affymetrix PrimeView™ arrays at the DanaFarber Cancer Institute Microarray Core.

FIG. 11 shows the levels of mRNA expression of various RaR subtypes in atamibarotene responsive (Au565) and non-responsive (HCC1143) cell linemeasured using the above protocol. Expression of RARA mRNA is 8-foldhigher in the responsive cell line versus the non-responsive cell line,while expression of RaR-ß and RaR-γ is not significantly differentbetween the cell lines. This confirms that RARA mRNA expression analysiscorrelates with RARA super enhancer strength and sensitivity to an RARAagonist, as well as demonstrating that RARA mRNA level can be used topredict sensitivity to such agonist.

II. RNA-Seq. RARA expression levels are quantified by RNA-Seq. Poly-ARNA-Seq is performed and reads are aligned to the HG19 transcriptomeusing RSEM software (parameters=--samtools-sort-mem 3G --ci-memory 3072--bowtie-chunkmbs 1024 --quiet --output-genome-bam --bowtie2--strand-specific) land then mRNA quantification is done using RSEM andreported in Transcripts Per Million (TPM). The plotted values show thelog2(FPKM+1) levels for RARA (y-axis) versus the super-enhancer score(RARA/MALAT1) for 48 primary AML patients (FIG. 12 ) have a SpearmanCorrelation of 0.589 and a R² value of 0.0457.

III. qPCR. Expression is also studied using rt-qPCR. Cells were countedand plated at 200,000 cells per mL for each cell line. Cells are thentreated with 500 nM tamibarotene or vehicle for 48 hours. RNA isextracted from cells using Trizol® and purified using the mirVana™ RNApurification kit (both from Life Technologies), following themanufacturer's protocol. The purified RNA is converted to cDNA usingSuperScript® Vilo™ reverse transcriptase (Life Technologies) followingthe manufacturer's protocol. Transcript abundance is then measured usingTaqman® probes (Life Technologies) to RARA and to 18s rRNA fornormalization. For RARA, probe Hs00940446 ml is used with an ampliconlength of 68 and spanning exons 6-7. For 18S rRNA probe 4319413E with a187 bp amplicon was used. The qPCR is run using Taqman® Gene ExpressionMaster Mix (Life Technologies) following the manufacturer's protocol.Analysis of the data is performed using the delta Ct method. The 18srRNA Ct value is subtracted from each sample to normalize to cellabundance. The dCt values indicate relative fold differences in RNAtranscript abundance. Lower dCt values indicate higher mRNA expression.The results for rt-qPCR values for 6 different breast cancer cell linesgraphed in order of their SE strength (from highest (SKBR3) to lowest(HCC1143) is shown in FIG. 13 . As can be seen rt-qPCR value correlatewell with super enhancer strength at the RARA locus. FIGS. 14A-B showthat the dCT values have a high correlation with SE strength and withsensitivity as measured by EC₅₀.

IV. Western Blotting. Cells are counted and adjusted to 2 million percell line and pelleted. Cell pellets are lysed using RIPA buffer (LifeTechnologies) plus Roche protease cocktail in a 1004, volume. Samplesare cleared by centrifugation at 20,000×g for 5 minutes. Ten μL ofsample is loaded on to 4-12% Bis-Tris gels (Life Technologies).Antibodies used are Abcam 1791 histone H3 for loading control and SigmaAldrich SAB1404298 for RARA. The results are shown in FIG. 15 . Theredoes not appear to be a good correlation between RARA protein levels andeither size of the super-enhancer or sensitivity of the cells to an RARAagonist. This confirms the prior results of another group that alsodemonstrated a lack of correlation between RARA protein expression andretinoid sensitivity (G Paroni et al., Oncogene, 31:3431-43 (2012); seeFIG. 1C in reference).

V. Z-score from TCGA. Z-scores of expression from the TCGA datasets areobtained from the cBio Portal for Cancer Genomics (which can be accessedon the world-wide web; see cbioportal.org/public-portal/).

VI. MDS expression measurements. Raw Affymetrix expression data of 159MDS patients and 17 normals associated with Gerstung et. al (PMID:25574665) are downloaded from the NCBI Gene Expression Omnibus(GSE58831). The expression data is normalized in R using the “justRMA”function with default parameters to produce normalized probe-levelvalues across all the samples. The value of the Affymetrix probe‘203749_s_at’ is used to represent the RARA mRNA level. The mean andstandard deviation of RARA levels in the 17 normal samples is calculatedand then used to generate RARA mRNA Z-scores.

Example 4. RARA Gene Copy Number is not Correlated with Sensitivity toTamibarotene

In order to rule out the possibility that sensitivity to tamibarotenewas based on HER2 gene copy number and that RARA and HER2 amplificationwere co-dependent, as suggested by G. Paroni et al., supra., we analyzethe copy number of the RARA and HER2 genes in two responsive breastcancer cell lines—Au565 and T47D. DNA is extracted using the QlAamp® DNAMini Kit (Qiagen) following the manufacturer's instructions. Thepurified DNA is analyzed using a Human Cytoscan® HD microarray fromAffymetrix. The data is processed using the package Aroma fromBioconductor in the statistical environment R, and graphed using base Rgraphics. As shown in FIGS. 16A-B, while the Au565 cell line has ahigher HER2 gene copy number (Box 1) than RARA (Box 2), the same is notseen in the equally tamibarotene-responsive T47D line. Moreover, thereis little difference in RARA gene copy number between the two celllines. This demonstrates that amplification of RARA gene copy number isnot dependent upon HER2 copy number and that sensitivity to an RARAagonist is not dependent upon HER2 copy number.

Example 5. Identification of RARA Associated Super Enhancers in OtherCancers

We use the known location of the RARA super enhancer domain to probeadditional patient cancer samples to determine if such cancers had aRARA super enhancer. We found a large super enhancer associated withRARA in both glioblastoma and neuroblastoma patient samples, but not incolorectal cancer samples (see FIGS. 17A-17C). This suggests thatstratification of patients for treatment with a RARA agonist can also becarried out in patients suffering from either glioblastoma orneuroblastoma. Moreover, Z-score analysis of RARA mRNA levels suggestthat certain subjects suffering from colorectal cancer may be responsiveto a RARA agonist, despite these super-enhancer results (see FIG. 30 ).

Example 6. Sensitivity of a HCC1954 Breast Cancer Cell Xenograft toTamibarotene

Breast cancer cell line (HCC1954)-derived xenograft models in BALB/cnude immunocompromised mice are prepared by Crown Biosciences (Beijing,China) essentially as follows.

Six to eight week-old BALB/c nude immunocompromised mice, weighingbetween 18-20 g are inoculated subcutaneously at the right flank regionwith HCC1954 tumor cells (5×10⁶) in 0.1 ml of PBS (1:1 matrigel) fortumor development. When the mean tumor size reaches a volume between100-200 mm³ animals are matched by tumor volume into treatment groups tobe used for dosing and dosing initiated. Tamibarotene is administeredorally in pH 8 adjusted PBS, 1% DMSO on a daily schedule (QD, PO) for upto 21 days. The final dosage in mice is 6 mg per kg per day in the highdose arm (n=10) and 3 mg per kg per day in the low dose arm (n=10) in a10 ml/kg volume. Mice in the vehicle arm (n=10) are given the sameschedule, volume, and formulation but lacking drug. Tumor volume ismeasured twice weekly by caliper measurement.

The results of these experiments demonstrate that tamibarotene reducestumor volume in this model in a dose-dependent manner (FIG. 18 ). Thereduction in tumor volume is just below statistical significance in thelow-dose arm (p=0.0552) and statistically significant in the high dosearm (p=0.0048) by t-test using Welch's correction. The results in thisxenograft model confirm the sensitivity of HCC1945 cells in culture totamibarotene.

Example 7. RARA Super Enhancer Strength Ordinal Rank Cutoff in BreastCancer

The total enhancer/super enhancer profile of one-hundred seventy breastcancer samples (both patient samples and breast cancer cell lines,including HCC1954) are analyzed using H3K27Ac and ChIP-Seq as describedin Example 1. In each of the samples, the ordinal rank of theRARA-associated enhancer in terms of strength (as measured by H3K27Ac)is determined as compared to other enhancers and super-enhancers in thesame cell and the determined ordinal ranks plotted on a rank-order bargraph (FIG. 19 ). In HCC1954 it was determined that the RARA superenhancer was the 204^(th) strongest enhancer in that cell (see FIG. 19). Based on this result and the responsiveness of HCC1954 totamibarotene, we set the ordinal cutoff for potentialtamibarotene-responsive breast cancer patients to those who have a RARAsuper enhancer in their tumor that is at least the 200^(th) strongest.As determined from our analysis of 48 primary breast cancer tumor cellsamples from human subjects, 55.3% of those samples had a RARA superenhancer that was at least the 200^(th) strongest in those cells (FIG.20A). Therefore, we set the prevalence cutoff at 55.3%. That sameprevalence cutoff is also used as the RARA mRNA prevalence cutoff whenidentifying potential breast cancer responders to tamibarotene based onRARA mRNA measurements.

When the primary breast cancer samples are further broken down bysubtype, different prevalence cutoffs for each subtype are generatedusing the same RARA super enhancer ordinal cutoff of 200. Theseprevalence cutoffs are 78.6% for hormone receptor-positive; 56.3% forHER2 positive; and 35.2% for triple negative breast cancer (FIG. 20B).

Example 8. RARA mRNA Levels Correlate with Responsiveness toTamibarotene in Breast Cancer PDXs

Low Passage TumorGraft® models of human breast cancer inimmunocompromised female mice (Harlan; nu/nu nude) are created byChampions Oncology (Baltimore, Maryland) using the following protocol.

Stock mice are implanted with patient breast cancer tumor samples (n=3for each separate patient sample and for the controls), which areallowed to grow to 1-1.5 cm³. Tumors are then harvested from stock miceand fragments thereof are re-implanted unilaterally on the left flank ofpre-study mice (female; Harlan; nu/nu nude, 5-8 weeks of age; n=3 foreach separate patient sample and for the controls). When tumors reachapproximately 100-300 mm³, pre-study animals are matched by tumor volumeinto treatment groups to be used for dosing and dosing initiated on Day0. Tamibarotene is administered orally in pH 8 adjusted PBS, 1% DMSO ona daily schedule at a final dose of 6 mg per kg body weight in a 10ml/kg volume. Mice in the vehicle arm are given the same schedule,volume, and formulation, but lacking tamibarotene.

Tumor volumes are measured by caliper twice a week. A final tumor volumemeasurement is taken on the last day of treatment. FIGS. 21A-B show theresponsiveness of two of these PDXs (CTG-1059 (high RARA mRNA) andCTG-0012 (low RARA mRNA) to tamibarotene (“SY1425”). CTG-1059demonstrates a statistically significant decrease in tumor volume after25 days of tamibarotene treatment as compared to a vehicle only control.CTG-0012 did not show any significant difference between tamibaroteneand the control.

RARA mRNA levels in both of these PDXs, as well as 7 other breast cancerPDXs are determined using RNA-seq as described in Example 3. We assumethat these 9 PDXs represent a population having a normal distribution ofRARA mRNA levels and using the 55.3% prevalence cutoff as determinedbased on RARA SE strength ordinal, the tamibarotene-responsive CTG-1059has a RARA mRNA level prevalence value above 55.3%, while thenon-responsive CTG-0012 has a RARA mRNA level prevalence value below55.3% (FIG. 22 ).

Example 9. RARA Super Enhancer Strength Ordinal Rank Cutoff in AML

The total enhancer/super enhancer profile of 95 AML samples (bothpatient samples and AML cell lines, including SigM5, MV411, HEL andKasumi) are analyzed using H3K27Ac and ChIP-Seq as described inExample 1. In each of the samples, the ordinal rank of theRARA-associated enhancer in terms of strength (as measured by H3K27Ac)is determined as compared to other enhancers and super-enhancers in thesame cell and the determined ordinal ranks are plotted on a rank-orderbar graph (FIGS. 23A-C). In MV411, it was determined that theRARA-associated enhancer was the 133^(rd) strongest enhancer. MV411 isthe confirmed tamibarotene-responsive cell line having the lowest superenhancer strength ordinal. In HEL, it was determined that theRARA-associated enhancer was the 155^(th) strongest enhancer. HEL is theconfirmed tamibarotene non-responsive cell line having the highest superenhancer strength ordinal. Based upon these values, we set the RARAenhancer strength ordinal cutoff at 150, a value in between the HELordinal and the MV411 ordinal.

As determined from our analysis of 70 primary AML cell samples fromhuman subjects, 36% of those samples had a RARA super enhancer that wasat least the 150^(th) strongest in those cells (FIG. 24 ). Therefore, weset the prevalence cutoff at 36%. That same prevalence cutoff is alsoused as the RARA mRNA prevalence cutoff when identifying potential AMLresponders to tamibarotene based on RARA mRNA measurements.

We also quantified the enhancers for an expanded panel of AML cell linesby ratio of RARA enhancer to MALAT1 enhancer. Plotting this enhancerstrength ratio value against sensitivity to tamibarotene, we confirmedthat 5 out of 6 cell lines bearing RARA enhancer strength ratios above 1are responsive, while only 4 out of 7 cell lines bearing enhancers belowthis level are responsive (FIG. 32 ). When the cutoff is moved toRARA/MALAT enhancer ratio of 1.4 or higher, all of the cell lines (4 outof 4) are responsive.

Example 10. RARA Super Enhancer Strength Ordinal Rank Cutoff in AMLCorrelates with RARA mRNA Levels

The AML patient samples used to determine the 36% RARA super enhancerstrength ordinal prevalence cutoff, are binned into two groups—thosehaving a prevalence rank of 36% or higher (i.e., a lower % value) andthose having a prevalence rank lower than 36% (i.e., a higher %value)—and assayed for RARA mRNA level using RNA-seq as described inExample 3. The results are shown in FIG. 25 . The group at or higherthan the 36% prevalence rank in RARA super enhancer strength ordinal hasa statistically significant higher level of RARA mRNA than the groupbelow the prevalence rank (p<0.001). This again confirmed that aprevalence cutoff determined at the super-enhancer level can also beused as the prevalence cutoff at the mRNA level.

We also determined the RARA mRNA levels in 11 different AML cell linesusing RNA-seq and compared the mRNA levels to sensitivity totamibarotene. The tested AML cell lines partitioned into two distinctgroups based on their sensitivity or insensitivity to tamibarotene.Tamibarotene-sensitive cell lines all had RARA mRNA measured byRNAseq >10 TPM, while three insensitive cell lines had levels below thiscut-off level (FIG. 33 ).

Example 11. Sensitivity of Various AML Patient Sample-derived Xenograftsto Tamibarotene

Different AML patient sample (AM8096, AM5512, AM7577 and AM7440)-derivedxenograft models in BALB/c nude immunocompromised mice are prepared byCrown Biosciences (Beijing, China) essentially as follows.

Approximately 2×10⁶ cells from each patient sample are suspended in 100μL PBS and injected into separate mice (n=3 for each different patientsample and for the control) by IV tail injection. For AM5512, AM7577 andAM7440 xenografts, tumor burden is considered high enough to starttreatment when the concentration of human CD45⁺ cells reaches ˜1-5% inthe animal's peripheral blood. Human CD45⁺ cells are detected in mouseblood (obtained via eye bleeds) using a fluorescence activated cellsorter and FITC anti-human CD45 (Biolegend, Cat# 304037). For AM8096xenografts, treatment is begun 40 days after injection of cells.

Tamibarotene is administered orally in pH 8 adjusted PBS, 1% DMSO on adaily schedule at a final dose of 6 mg per kg body weight in a 10 ml/kgvolume. Mice in the vehicle arm are given the same schedule, volume, andformulation, but lacking tamibarotene. Human CD45⁺ cell levels inperipheral blood from the treated animals and control animals aremeasured once a week.

AM5512 and AM8096 xenografts show a significant reduction in the total %of CD45⁺ cells, as well as in the % of CD45⁺ cells in blood, bone marrowand spleen, when treated with tamibarotene as compared to the vehiclecontrol after 35 days of treatment (FIGS. 26A-F). On the other hand,AM7577 and AM7440 show no significant reduction in tumor volume betweenthe tamibarotene treated and vehicle treated animals either overall orin any of blood, bone marrow or spleen (FIGS. 27A-D).

FIG. 28 shows a rank ordering of RARA mRNA levels from individualpatient samples used in the binning analysis shown in FIG. 25 . Inaddition, FIG. 28 includes the RARA mRNA levels of each of the patientsamples used in the xenograft study. The two non-responders in thexenograft study, AM7577 and AM7440, have RARA mRNA levels that fall wellbelow the 36% prevalence cutoff. One of the responders, AM8096, has aRARA mRNA level that is above the 36% prevalence cutoff. The otherresponder, AM5512, falls slightly below the 36% prevalence cutoff (46.9%prevalence value). These RARA mRNA results suggest that the prevalencecutoff could be adjusted downward (e.g., to 46.9%) to maximize thenumber of potential responders.

Example 12. AM5512 is Sensitive to Tamibarotene but not ATRA

AM5512 xenograft mice are prepared as described above. When theconcentration of human CD45⁺ cells reach ˜1-5% in the animal'speripheral blood, they are treated with either tamibarotene (n=7; 3mg/kg, BID), all-trans retinoic acid (ATRA; n=7; 4 mg/kg BID) or avehicle control (n=5). While tamibarotene is a RARA-specific agonist,ATRA agonizes all retinoic acid receptors (RAR-α, RAR-ß and RAR-γ). Asshown in FIGS. 29A-B, AM5512 xenograft mice treated with tamibaroteneshow a significant reduction in % CD45⁺ cells after 28 days of treatmentas compared to both vehicle control and ATRA-treated animals and 5 ofthe 7 animals survive the course of the experiment. Surprisingly, whileAM5512 xenograft mice treated with ATRA show a reduction in % CD45⁺cells as compared to the vehicle control after 14 days of treatment,none of those mice survived past day 21.

Example 13. Subsets of Patient Samples with Other Cancers have HighLevels of RARA mRNA

For a number of different cancer types, we use RARA mRNA level z-scoresprovided by TCGA as described in Example 3. In a normal distribution ofa population, 5% of samples should have a RARA mRNA level that isgreater than 2 standard deviations from the mean. FIG. 30 is a tableshowing those specific cancer types having greater than 5% of sampleswith RARA mRNA levels greater than 2 standard deviations from the mean.Without being bound by theory, we believe that each of these cancerswill be susceptible to the stratification and treatment methods setforth herein.

We also look specifically at samples from patients suffering frommyelodysplastic syndrome, which is believed to be a cancer closelyrelated to AML. RARA mRNA levels from 176 patients (17 normal; 159 MDS)are obtained as described in Example 3. The samples are binned based ondisease state and RARA mRNA levels plotted as shown in FIG. 31 .Statistical analysis of the results (T-test) show that RARA mRNA levelsare significantly elevated in MDS patients samples as compared to normalpatient samples (p=0.08751).

To further validate the presence of elevated RARA in MDS, ChIP-seqH3K27ac enhancer analysis was performed on bone marrow samples collectedfrom two MDS patients. For both of these patients, the RARA gene locushad a markedly stronger H3K27ac signal in the two MDS patient bonemarrow samples, compared to the signal found in the blast cells ofhealthy donors. The strength of the RARA super-enhancer in the MDSpatients was similar to that in blasts from the subset of AML patientsthat have strong RARA super-enhancers and high levels of RARA mRNA.Furthermore, the RARA super-enhancer in the MDS patient cells had a highRARA enhancer strength ordinal (9 and 60, respectively) compared toblast and immature cell types from healthy subjects.

Without being bound by theory, we believe that MDS patients will also besusceptible to the stratification and treatment methods set forthherein.

Equivalents and Scope

In the embodiments articles such as “a,” “an,” and “the” may mean one ormore than one unless indicated to the contrary or otherwise evident fromthe context. Embodiments or descriptions that include “or” between oneor more members of a group are considered satisfied if one, more thanone, or all of the group members are present in, employed in, orotherwise relevant to a given product or process unless indicated to thecontrary or otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed embodiments isintroduced into another embodiment. For example, any embodiment that isdependent on another embodiment can be modified to include one or morelimitations found in any other embodiment that is dependent on the samebase embodiment. Where elements are presented as lists, e.g., in Markushgroup format, each subgroup of the elements is also disclosed, and anyelement(s) can be removed from the group. It should it be understoodthat, in general, where the invention, or aspects of the invention,is/are referred to as comprising particular elements and/or features,certain embodiments of the invention or aspects of the inventionconsist, or consist essentially of, such elements and/or features. Forpurposes of simplicity, those embodiments have not been specifically setforth in haec verba herein. It is also noted that the terms “comprising”and “containing” are intended to be open and permits the inclusion ofadditional elements or steps. Where ranges are given, endpoints areincluded. Furthermore, unless otherwise indicated or otherwise evidentfrom the context and understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value orsub-range within the stated ranges in different embodiments of theinvention, to the tenth of the unit of the lower limit of the range,unless the context clearly dictates otherwise.

This application refers to various issued patents, published patentapplications, journal articles, and other publications, all of which areincorporated herein by reference. If there is a conflict between any ofthe incorporated references and the instant specification, thespecification shall control. In addition, any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the embodiments. Because suchembodiments are deemed to be known to one of ordinary skill in the art,they may be excluded even if the exclusion is not set forth explicitlyherein. Any particular embodiment of the invention can be excluded fromany embodiment, for any reason, whether or not related to the existenceof prior art.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended embodiments. Those ofordinary skill in the art will appreciate that various changes andmodifications to this description may be made without departing from thespirit or scope of the present invention, as defined in the followingembodiments.

1. A method of selecting and treating a human subject suffering fromnon-acute promyelocytic leukemia acute myelogenous leukemia (non-APLAML), the method comprising: a. selecting the subject if a sample ofdiseased cells from the subject has a level of retinoic acid receptoralpha (RARA) mRNA equal to or above a predetermined threshold, whereinthe RARA mRNA is transcribed from a RARA gene that encodes a functionalRARA gene and specifically excludes gene fusions that comprise all or aportion of the RARA gene; and b. administering to the selected subjectan amount of tamibarotene effective to treat the disease. 2-12.(canceled)